Anti-CXCR1 compositions and methods

ABSTRACT

The present invention provides methods of treating cancer by administering an IL8-CXCR1 pathway inhibitor (e.g., an anti-CXCR1 antibody or Repertaxin) alone or in combination with an additional chemotherapeutic agent such that non-tumorigenic and tumorigenic cancer cells in a subject are killed. The present invention also provides compositions and methods for detecting the presence of and isolating solid tumor stem cells in a patient (e.g., based on the presence of CXCR1 or FBXO21).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/616,579, filed Nov. 11, 2009, which claims priority to U.S.Provisional Patent Application No. 61/113,458, filed Nov. 11, 2008, eachof which are hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA129765, CA101860and CA046592 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention provides methods of treating cancer byadministering an IL8-CXCR1 pathway inhibitor (e.g., an anti-CXCR1antibody or Repertaxin) alone or in combination with an additionalchemotherapeutic agent such that non-tumorigenic and tumorigenic cancercells in a subject are killed. The present invention also providescompositions and methods for detecting the presence of and isolatingsolid tumor stem cells in a patient (e.g., based on the presence ofCXCR1 or FBXO21).

BACKGROUND

Cancer remains the number two cause of mortality in this country,resulting in over 500,000 deaths per year. Despite advances in detectionand treatment, cancer mortality remains high. Despite the remarkableprogress in understanding the molecular basis of cancer, this knowledgehas not yet been translated into effective therapeutic strategies.

In particular, breast cancer is the most common cancer in Americanwomen, with approximately one in nine women developing breast cancer intheir lifetime. Unfortunately, metastatic breast cancer is still anincurable disease. Most women with metastatic breast cancer succumb tothe disease.

Traditional modes of therapy (radiation therapy, chemotherapy, andhormonal therapy), while useful, have been limited by the emergence oftreatment-resistant cancer cells. Clearly, new approaches are needed toidentify targets for treating metastatic breast cancer and cancergenerally.

SUMMARY OF THE INVENTION

The present invention provides methods of treating cancer byadministering an IL8-CXCR1 pathway inhibitor (e.g., an anti-CXCR1antibody or Repertaxin) alone or in combination with an additionalchemotherapeutic agent such that non-tumorigenic and tumorigenic cancercells in a subject are killed. The present invention also providescompositions and methods for treating and diagnosing the presence ofsolid tumor stem cells in a patient (e.g., based on the presence ofCXCR1 or FBXO21).

In some embodiments, the present invention provides methods of treatingcancer comprising: administering an IL8-CXCR1 pathway antagonist and anadditional chemotherapeutic agent to a subject. In certain embodiments,the present invention provides methods of reducing or eliminating cancerstem cells and non-tumorigenic cancer cells in a subject comprising:administering Repertaxin or derivative thereof to a subject underconditions such that at least a portion of the cancer stem cells and atleast a portion of the non-tumorigenic cancer cells are killed. In otherembodiments, the present invention provides methods of reducing oreliminating cancer stem cells and non-tumorigenic cancer cells in asubject comprising: administering an IL8-CXCR1 pathway antagonist and anadditional chemotherapeutic agent to a subject under conditions suchthat at least a portion of the cancer stem cells and at least a portionof the non-tumorigenic cancer cells are killed. In particularembodiments, the present invention provides compositions or kitscomprising an IL8-CXCR1 pathway antagonist and an additionalchemotherapeutic agent.

In certain embodiments, the IL8-CXCR1 pathway antagonist comprises anagent that specifically blocks the binding of IL8 to CXCR1. In someembodiments, the agent binds to (is specific for) CXCR1, but does notbind to CXCR2. In other embodiments, the agent binds to CXCR1. Inparticular embodiments, the agent comprises an anti-CXCR1 antibody orantibody fragment. In additional embodiments, the agent comprisesRepertaxin or a derivative thereof. In further embodiments, theadditional chemotherapeutic agent comprises an anti-mitotic compound. Incertain embodiments, the anti-mitotic compound is selected from thegroup consisting of: docetaxel, doxorubicin, paclitaxel, fluorouracil,vincristine, vinblastine, nocodazole, colchicine, podophyllotoxin,steganacin, and combretastatin. In other embodiments, the anti-mitoticcompound is a catharalthus alkaloids (e.g., vincristine andvinblastine); or a benzimidazole carbamates such as nocodazole; orcolchicine or related compounds such as podophyllotoxin, steganacin orcombretastatin; or a taxane such as paclitaxel and docetaxel. In certainembodiments, the additional chemotherapeutic agent comprises docetaxel.

In particular embodiments, the subject has a type of cancer that, whentreated with a chemotherapeutic, has increased levels of IL-8 production(e.g., which causes an increase in cancer stem cell number of motility).In some embodiments, the subject has a type of cancer selected from thegroup consisting of: prostate cancer, ovarian cancer, breast cancer,melanoma, non-small cell lung cancer, small-cell lung cancer, andesophageal adenocarcinoma.

In other embodiments, the present invention provides methods ofdetecting solid tumor stem cells comprising; a) providing: i) a sampletaken from a tumor of a subject, and ii) an antibody, or antibodyfragment (or other binding molecule), specific for the CXCR1 protein orFBXO21 protein (or another protein from Table 1); and b) contacting thetissue sample with the antibody, or antibody fragment, under conditionssuch that the presence or absence of CXCR1+ or FBXO21+ solid tumor stemcells are detected.

In particular embodiments, the antibody, or antibody fragment, isconjugated to a signal molecule. In further embodiments, the signalmolecule comprises a fluorescent molecule. In other embodiments, thesignal molecule comprises an enzyme that can catalyze a color producingreaction in the presence of a colorimetric substrate. In certainembodiments, the method further comprises contacting the sample with asecondary antibody, or secondary antibody fragment, specific for theantibody or antibody fragment. In other embodiments, the secondaryantibody, or secondary antibody fragment, comprises a signal molecule.In particular embodiments, no other proteins or nucleic acids areassayed in order to determine the presence or absence of the CXCR1 orFBXO21+ solid tumor stem cells. In additional embodiments, the tumor isselected from the group consisting of: a prostate cancer tumor, anovarian cancer tumor, a breast cancer tumor, a melanoma, a non-smallcell lung cancer tumor, a small-cell lung cancer tumor, and anesophageal adenocarcinoma tumor.

In some embodiments, the present invention provides methods of enrichingfor a population of solid tumor stem cells comprising: a) disassociatinga solid tumor to generate disassociated cells; b) contacting thedisassociated cells with a reagent that binds CXCR1 or FBXO21 (or otherprotein from Table 1); and c) selecting cells that bind to the reagentunder conditions such that an a population enriched for solid tumor stemcells is generated.

In certain embodiments, no additional reagents are employed in order togenerate the population enriched for solid tumor stem cells. In someembodiments, the tumor is selected from the group consisting of: aprostate cancer tumor, an ovarian cancer tumor, a breast cancer tumor, amelanoma, a non-small cell lung cancer tumor, a small-cell lung cancertumor, and an esophageal adenocarcinoma tumor. In further embodiments,the reagent is an antibody or antibody fragment (e.g., Fab fragment). Inadditional embodiments, the reagent is conjugated to a fluorochrome ormagnetic particles. In other embodiments, the selecting cells isperformed by flow cytometry, fluorescence activated cell sorting,panning, affinity column separation, or magnetic selection.

In particular embodiments, the present invention provides an enrichedpopulation of solid tumor stem cells isolated by the methods describedherein.

In some embodiments, the present invention provides isolated populationsof cancer stem cells that are: a) tumorigenic; and b) CXCR1+ or FBXO21+.In certain embodiments, the cancer stem cells are cancer stem cellsselected from the group consisting of: prostate cancer stem cells,ovarian cancer stem cells, breast cancer stem cells, skin cancer stemcells, non-small cell lung cancer stem cells, small-cell lung cancerstem cells, and esophageal adenocarcinoma stem cells. In otherembodiments, the population comprises at least 60% cancer stem cells andless than 40% non-tumorigenic tumor cells. In further embodiments, thecancer stem cells: are enriched at least two-fold compared tounfractionated non-tumorigenic tumor cells (e.g., 2-fold, 3-fold,4-fold, 5-fold, . . . , 10-fold, . . . 100-fold, . . . 1000-fold).

In some embodiments, the present invention provides methods forobtaining from a tumor a cellular composition comprising cancer stemcells and non-tumorigenic tumor cells, wherein at least 60% aretumorigenic stem cells and 40% or less are non-tumorigenic tumor cells,the method comprising: a) obtaining a dissociated mixture of tumor cellsfrom a tumor; b) separating the mixture of tumor cells into a firstfraction comprising at least 60% cancer stem cells and 40% or lessnon-tumorigenic tumor cells and a second fraction of tumor cellsdepleted of cancer stem cells wherein the separating is by contactingthe mixture with a reagent against CXCR1 or FBXO21; and c) demonstratingthe first fraction to be tumorigenic by: i) serial injection into afirst host animal and the second fraction to be non-tumorigenic byserial injection into a second host animal. In certain embodiments, theseparating is performed by flow cytometry, fluorescence activated cellsorting (FACS), panning, affinity chromatography or magnetic selection.In some embodiments, the separating is performed by fluorescenceactivated cell sorters (FACS) analysis.

In particular embodiments, the present invention provides methods forselecting a treatment for a patient having a solid tumor, comprising:(a) obtaining a sample from the patient; (b) identifying the presence ofCXCR1+ or FBXO21+ solid tumor stem cell in the sample; and (c) selectinga treatment for the patient that targets CXCR1+ or FBXO21+ solid tumorstem cells (e.g., selecting the use of an anti-CXCR1 antibody orantibody fragment). In certain embodiments, the CXCR1+ or FBXO21+ solidtumor stem cells are cancer stem cells selected from the groupconsisting of: prostate cancer stem cells, ovarian cancer stem cells,breast cancer stem cells, skin cancer stem cells, non-small cell lungcancer stem cells, small-cell lung cancer stem cells, and esophagealadenocarcinoma stem cells.

In some embodiments, the present invention provides methods forscreening a compound, comprising: a) exposing a sample comprising aCXCR1+ or FBXO21+ cancer stem cell to a candidate anti-neoplasticcompound, wherein the candidate anti-neoplastic compound comprises aCXCR1 or FBXO21 antagonist or a IL8-CXCR1 signaling pathway antagonist;and b) detecting a change in the cell in response to the compound.

In certain embodiments, the sample comprises a non-adherent mammosphere.In further embodiments, the CXCR1 or FBXO21 antagonist, or IL8-CXCR1signaling pathway antagonist comprises an antibody or antibody fragment.In some embodiments, the CXCR1 antagonist is a derivative of Repartaxin.In other embodiments, the detecting comprises detecting cell death ofthe tumorigenic breast cell. In further embodiments, the methods furthercomprise identifying the candidate anti-neoplastic agent as capable ofkilling tumorigenic cells as well as non-tumorigenic cancer cells.

In some embodiments, the present invention provides methods fordetermining the capability of a test compound to inhibit tumorigenesisof solid tumor stem cells comprising: a) obtaining enriched solid tumorstem cells, wherein the solid tumor stem cells: i) are enriched at leasttwo-fold compared to unfractionated tumor cells; and ii) express CXCR1or FBXO21; b) exposing a first set, but not a second set, of the solidtumor stem cells to a test compound; c) injecting the first set of thesolid tumor stem cells into a first host animal and injecting the secondset of solid tumor stem cells into a second host animal; and d)comparing a tumor, if present, in the first animal with a tumor formedin the second animal in order to determine if the test compound inhibitstumor formation. In particular embodiments, the test compound is a CXCR1or FBXO21 inhibitor, or a IL8-CXCR1 inhibitor pathway inhibitor.

In further embodiments, the present invention provides methods fordetermining the capability of a test compound to inhibit tumorigenesisof solid tumor stem cells comprising: a) obtaining a sample comprisingat least 60% solid tumor stem cells, wherein the solid tumor stem cellsexpress CXCR1 or FBXO21; b) injecting the solid tumor stem cells intofirst and second host animals; c) treating the first host animal with atest compound, and not treating the second host animal with the testcompound; and d) comparing a tumor, if present, in the first animal witha tumor formed in the second animal in order to determine if the testcompound inhibits tumor formation. In other embodiments, the testcompound is a CXCR1 or FBXO21 inhibitor or an IL8-CXCR1 pathwayinhibitor.

DESCRIPTION OF FIGURES

FIG. 1 shows the ALDEFLUOR-positive cell populations from breast cancercell lines (MDA-MB-453, SUM159) have cancer stem cell properties. A-B,G-H. Representative flow cytometry analysis of ALDH enzymatic activityin MDA-MB-453 (A-B) and SUM159 cells (G-H). The ALDEFLUOR assay wasperformed as described in Example 1 below. (C, I) The ALDEFLUOR-positivepopulation was capable of generating tumors in NOD/SCID mice whichrecapitulated the phenotypic heterogeneity of the initial tumor. (F, L)Tumor growth curves were plotted for different numbers of cells injected(for MDA-MB-453: 50,000 cells, 5,000 cells, and 500 cells and forSUM159: 100,000 cells, 10,000 cells, and 1,000 cells) and for eachpopulation (ALDEFLUOR-positive, ALDEFLUOR-negative, unseparated). Tumorgrowth kinetics correlated with the latency and size of tumor formationand the number of ALDEFLUOR-positive cells (F, L). (D, J) H&E stainingof ALDEFLUOR-positive cells' injection site, revealing presence of tumorcells (D: MDA-MB-453 ALDEFLUOR-positive cells' injection site, and J:SUM59 ALDEFLUOR-positive cells' injection site). (E, K) TheALDEFLUOR-negative cells' injection site contained only residualMatrigel, apoptotic cells, and mouse tissue (E: MDA-MB-453ALDEFLUOR-negative cells' injection site, and K: SUM59ALDEFLUOR-negative cells' injection site). Data represent mean±SD.

FIG. 2 shows classification of the ALDEFLUOR-positive andALDEFLUOR-negative populations isolated from breast cell lines based onthe “cancer stem cell signature”. FIG. 2A. Hierarchical clustering of 16samples based on a 413-gene expression signature. Each row of the datamatrix represents a gene and each column represents a sample. Note theseparation between ALDEFLUOR-positive (underlined names) and negativesamples (non-underlined names) with the 413 genes for 15 out of the 16samples. Some genes included in the signature are referenced by theirHUGO abbreviation as used in ‘Entrez Gene’ (Genes down-regulated in theALDEFLUOR-positive populations are labeled in green and genesup-regulated in the ALDEFLUOR-positive populations are labeled in red).FIG. 2B-C. To confirm the gene expression results, in a set of fivebreast cancer cell lines sorted for the ALDEFLUOR phenotype, theexpression of five discriminator genes overexpressed inALDEFLUOR-positive populations (CXCR1/IL8RA, FBXO21, NFYA, NOTCH2 andRAD51L1) were measured by quantitative RT-PCR. The quantitative RT-PCRexpression levels of CXCR1 and FBXO21 are presented in this figure. Geneexpression levels measured by quantitative RT-PCR confirm the resultsobtained using DNA microarrays with an increase of CXCR1 and FBXO21 mRNAlevel in the ALDEFLUOR-positive population compared to theALDEFLUOR-negative population (p<0.05).

FIG. 3 shows the role of the IL8/CXCR1 axis in the regulation of breastcancer stem cells. A. Cells expressing CXCR1 are contained in theALDEFLUOR-positive population. The ALDEFLUOR-positive and -negativepopulation from four different breast cell lines (HCC1954, SUM159,MDA-MB-453, BrCa-MZ-01) were isolated by FACS, fixed, and analyzed forthe expression of CXCR1 protein by immunostaining and FACS analysis.ALDEFLUOR-positive cells were highly enriched in CXCR1-positive cellscompared to the ALDEFLUOR-negative population. B. Effect of IL8treatment on tumorosphere formation of three different cell lines(HCC1954, SUM159, MDA-MB-453). IL8 treatment increased the formation ofprimary and secondary tumorospheres in a dose-dependent manner. C.Effect of IL8 treatment on the ALDEFLUOR-positive population of fourdifferent cell lines cultured in adherent conditions. IL8 increased theALDEFLUOR-positive population in a dose-dependent manner in each of thefour cell lines analyzed (* p<0.05/** p<0.01, statistically significantdifferences from the control group).

FIG. 4 shows ALDEFLUOR-positive cells display increased metastaticpotential. A. The IL8/CXCR1 axis is involved in cancer stem cellinvasion. The role of the IL8/CXCR1 axis in invasion was assessed by aMatrigel invasion assay using serum or IL8 as attractant for threedifferent cell lines (HCC1954, MDA-MB-453, SUM159). ALDEFLUOR-positivecells were 6- to 20-fold more invasive than ALDEFLUOR-negative cells(p<0.01). When using IL8 (100 ng/ml) as attractant, it was observed thata significant increase of ALDEFLUOR-positive cells were invading throughMatrigel compared to serum as attractant (p<0.05). In contrast IL8 hadno effect on the invasive capacity of the ALDEFLUOR-negative population.B-M. The ALDEFLUOR-positive population displayed increased metastaticpotential. B-D. Quantification of the normalized photon flux measured atweekly intervals following inoculation of 100,000 luciferase infectedcells from each group (ALDEFLUOR-positive, ALDEFLUOR-negative,unseparated). E-J Detection of metastasis utilizing the bioluminescenceimaging software (E, G, I: Mice facing down; F, H, J: Mice facing up).Mice inoculated with ALDEFLUOR-positive cells developed severalmetastasis localized at different sites (bone, muscle, lung, softtissue) and displayed a higher photon flux emission than mice inoculatedwith unseparated cells, which developed no more than one metastasis permouse. In contrast, mice inoculated with ALDEFLUOR-negative cellsdeveloped only an occasional small metastasis, which was limited tolymph nodes. K-M. Histologic confirmation, by H&E staining, ofmetastasis in bone (K), soft tissue (L) and muscle (M) resulting frominjection of ALDEFLUOR-positive cells.

FIG. 5 shows the effect of CXCR1 inhibition on tumor cells viability(FIG. 5A) as well as on cancer stem cell viability (FIG. 5B).

FIG. 6 shows that Repertaxin treatment induces a bystander effectmediated by the FAS/FAS ligand signaling, and specifically shows thatthe cell growth inhibition induced by the Repertaxin treatment waspartially rescued by the addition of a FAS antagonist and that the cellstreated with a FAS agonist displayed a similar cell growth inhibitionthan the cells treated with Repertaxin.

FIG. 7 shows the activation of FAK, AKT and FOXOA3 activation withoutRepertaxin treatment (7A) and in the presence of Repertaxin (7B).

FIG. 8 shows the effect of Repertaxin, docetaxel, or the combinationthereof on one breast cancer cell line (8A, SUM159) and three humanbreast cancer xenografts generated from different patients (8B, MC1; 8C,UM2; and 8D, UM3).

FIG. 9 shows the effect of Repertaxin, docetaxel, or the combinationtreatment on the cancer stem cell population as assessed by theALDEFLUOR assay on various cells lines including SUM159 (9A), MC1 (9B),UM2 (9C), UM3 (9D).

FIG. 10 shows the effect of Repertaxin, docetaxel or the combination onserial dilutions of primary tumors (10A. SUM159, 10B. MC1, 10C. UM2,10D. UM3) that were implanted in the mammary fat pad of secondaryNOD-SCID mice.

FIG. 11 shows that Repertaxin treatment reduces the metastatic potentialof SUM159 cell line. FIG. 11A shows a quantification of the normalizedphoton flux measured at weekly intervals following inoculation withintracardiac administered SUM 159 cells. Metastasis formation wasmonitored using bioluminescence imaging (11B: Mice treated with salinesolution; 11C: Mice treated with Repertaxin).

FIG. 12 shows representations of the overlap between theALDEFLUOR-positive subpopulation and the CXCR1-positive subpopulation(top) or CXCR2-positive subpopulation (bottom) of SUM159 cells. B-C.SUM159 cells were cultured in adherent conditions and treated withrepertaxin (100 nM) or two specific blocking antibodies for CXCR1 (10μg/ml) or CXCR2 (10 μg/ml). After three days, the effect on the cancerstem cell population was analyzed using the ALDEFLUOR assay (B) cellviability was accessed after five days of treatment using the MTT assay(C). A significant reduction of the ALDEFLUOR-positive population andcell viability was observed following treatment with repertaxin oranti-CXCR1 antibody. In contrast no significant effect was observed withanti-CXCR2 antibody. D. After 4 days of treatment, the number ofapoptotic cells was evaluated utilizing a TUNEL assay. 36% apoptoticcells (stained in green) were detected in repertaxin treated cellscompared to the controls where mostly viable cells (stained in blue)were present. E-F. To determine whether cell death was mediated via abystander effect. CXCR1-positive and CXCR1-negative populations wereflow sorted and each population treated with various concentrations ofrepertaxin (D). A decrease in cell viability in CXCR1-positive andunsorted populations were detected whereas no effect was observed in theCXCR1-negative population (E). Dialyzed conditioned medium (dCM) fromCXCR1-positive cells treated for three days with repertaxin was utilizedto treat sorted CXCR1-positive, CXCR1-negative, or unsorted populations.Serial dilutions of dialyzed conditioned medium were utilized (Control,dCM ¼, dCM ½, dCM ¾, dCM). After two days of treatment, cell viabilitywas evaluated utilizing the MTT assay. A massive decrease in cellviability was observed in both CXCR1-negative and unseparatedpopulations whereas no effect was observed in the CXCR1-positivepopulation (F).

FIG. 13 shows tumorigenicity of the ALDEFLUOR-positive/CXCR1-positiveand ALDEFLUOR-positive/CXCR1-negative cell populations from SUM159 cellline. A. Tumor growth curves were plotted for different numbers of cellsinjected (50,000 cells, 5,000 cells, 1,000 cells, and 500 cells) and foreach population (ALDEFLUOR-positive/CXCR1-positive,ALDEFLUOR-positive/CXCR1-negative). Both cell populations generatedtumors. Tumor growth kinetics correlated with the latency and size oftumor formation and the number of cells injected. B-C. Tumors generatedby the ALDEFLUOR-positive/CXCR1-positive population reconstituted thephenotypic heterogeneity of the initial tumor upon serial passageswhereas the ALDEFLUOR-positive/CXCR1-negative population gave rise totumors containing only ALDEFLUOR-positive/CXCR1-negative cells. Wetransplanted both cell population for three passages.

FIG. 14 shows the effect of CXCR1 blockade on tumorsphere formation.SUM159 and HCC1954 cells were cultured in adherent conditions andtreated for three days with repertaxin (100 nM), an anti-CXCR1 blockingantibody (10 μg/ml), or an anti-CXCR2 blocking antibody (10 μg/ml).After three days of treatment, cells were detached and cultured insuspension. The number of tumorspheres formed after 5 days of culturewere evaluated. Similar results were observed for the both cell lineswith a significant decrease in primary and secondary tumorosphereformation in the repertaxin and anti CXCR1-treated conditions comparedto controls. In contrast, anti-CXCR2 blocking antibody had no effect ontumorosphere formation.

FIG. 15 shows the effect of repertaxin treatment on cell viability ofSUM159, HCC1954, and MDA-MB-453 cell lines. Three different cell lines(SUM159, HCC1954, MDA-MB-453) were cultured in adherent conditions andtreated with repertaxin (100 nM). Cell viability was evaluated afterone, three, and five days of treatment using the MTT assay. A decreasein cell viability was observed after 3 days of treatment for SUM159 andHCC1954 cell line. However, repertaxin did not effect the viability ofMDA-MB 453 cells.

FIG. 16 shows the effect of CXCR1 blockade on the ALDEFLUOR-positivepopulation in vitro. A-B. HCC1954 (A) and MDA-MB-453 (B) cells werecultured in adherent conditions and treated with repertaxin (100 nM) ortwo specific blocking antibodies for CXCR1 (10 μg/ml) or CXCR2 (10μg/ml). After three days, the effect on the cancer stem cell populationwas analyzed using the ALDEFLUOR assay. For HCC1954, a significantreduction of the ALDEFLUOR-positive population and cell viability wasobserved following treatment with repertaxin or anti-CXCR1 antibody. Incontrast no significant effect was observed with anti-CXCR2 antibody(A). For MDA-MB-453, np any effect on the ALDEFLUOR-positive populationwas observed (B).

FIG. 17 shows repertaxin treatment induces a bystander effect mediatedby FAS/FAS-ligand signaling. A. To determine whether the bystanderkilling effect induced by the repertaxin treatment was mediated byFAS-ligand, the level of soluble FAS-ligand in the medium was measuredutilizing an ELISA assay. After 4 days of treatment, greater than afour-fold increase of soluble FAS-Ligand was detected in the medium ofcells treated with repertaxin compared to non-treated controls. B. Thelevel of FAS-ligand mRNA was measured by RT-PCR and confirmed theincrease of FAS-ligand production after treatment with repertaxin.Similar results were observed after 4 days of treatment with a FASagonist that activates FAS signaling, with a five-fold increase of theFAS-ligand mRNA compared to the control. C. SUM159 cells were culturedin adherent conditions and treated with repertaxin alone or incombination with an anti-FAS-ligand. Cell growth inhibition induced bythe Repertaxin treatment was partially rescued by addition ofanti-FAS-Ligand. Cells treated with a FAS agonist displayed similar cellgrowth inhibition to cells treated with repertaxin alone. D-E. Theeffect of repertaxin treatment alone or in combination with ananti-FAS-ligand and the treatment of a FAS-agonist on the CXCR1-positiveand ALDEFLUOR-positive population was analyzed. The massive decrease inthe CXCR1-positive and ALDEFLUOR-positive population induced byrepertaxin treatment was not rescued by the anti-FAS-ligand andtreatment with FAS-agonist produced a ten-fold and three-fold increasein the percent of the CXCR1-positive and ALDEFLUOR-positive population,respectively.

FIG. 18 shows the effect of FAS agonist on CXCR1-positive andCXCR1-negative cells. CXCR1-positive and CXCR1-negative populations wereflow sorted and each population treated with various concentrations ofFAS agonist. A decrease in cell viability in CXCR1-negative and unsortedpopulations were detected whereas no effect was observed in theCXCR1-positive population.

FIG. 19 shows analysis of CXCR1 protein expression in the normal breaststem/progenitor population and effect of IL-8 treatment on mammosphereformation. A. The ALDEFLUOR-positive and -negative population fromnormal breast epithelial cells isolated form reduction mammoplasties wasisolated by FACS, fixed, and analyzed for the expression of CXCR1protein by immunostaining and FACS analysis. ALDEFLUOR-positive cellswere highly enriched in CXCR1-positive cells compared to theALDEFLUOR-negative population. B-C. Effect of IL8 treatment onmammosphere formation. IL8 treatment increased the formation of primary(B) and secondary mammospheres (C) in a dose-dependent manner.

FIG. 20 shows the effect of repertaxin treatment on the normal mammaryepithelial cells. A. Normal mammary epithelial cells isolated fromreduction mammoplasties were cultured in adherent condition and treatedwith repertaxin (100 nM or 500 nM) or FAS agonist (500 ng/ml). Afterfive days of treatment cell viability was evaluated using MTT assay.Repertaxin treatment or the FAS agonist had no effect on the viabilityof normal mammary epithelial cells cultured in adherent conditions, evenwhen high concentrations of repertaxin (500 nM) were utilized. B. Thelevel of soluble FAS-ligand was evaluated by Elisa assay in the mediumof normal mammary epithelial cells treated with repertaxin. After 4 daysof treatment an increase of soluble FAS-ligand was detected in themedium from treated cells. C. Analysis of FAS/CD95 expression in thenormal mammary epithelial cells by FACS analysis. No FAS/CD95 expressionwas detected in the normal mammary epithelial cells cultured in adherentcondition. D. Effect of repertaxin treatment on mammosphere formation.Normal mammary epithelial cells were cultured in adherent condition andtreated during four, eight, eleven and fifteen days with repertaxin (100nM). After repertaxin treatment cells were detached and cultured insuspension. A significant decrease of mammosphere-initiating cells wasobserved in the repertaxin-treated condition.

FIG. 21 shows the effect of repertaxin treatment on FAK, AKT and FOXO3aactivation. To evaluate the effect of repertaxin treatment on CXCR1downstream signaling, two different viral constructs were utilized, oneknocking down PTEN expression via a PTEN-siRNA and the other leading toFAK overexpression (Ad-FAK). A. SUM159 control, SUM159 PTEN-siRNA, andSUM159 Ad-FAK cells were cultured in adherent conditions for two days inthe absence or presence of 100 nM repertaxin and the activation of theFAK/AKT pathway was accessed by western blotting. Repertaxin treatmentled to a decrease in FAK Tyr397 and AKT Ser473 phosphorylation whereasPTEN deletion and FAK overexpression blocked the effect of repertaxintreatment on FAK and AKT activity. B. Utilizing immunofluorescencestaining on CXCR1-positive cells, we confirmed that Repertaxin treatmentresults in a disappearance of phospho-FAK (membranous staining in red)and phospho-AKT expression (cytoplasmic staining in red).Immunofluorescence staining with an anti-FOXO3A revealed a cytoplasmiclocation of FOXO3a (in red) in the untreated cells whereas repertaxintreatment induced a re-localization of FOXO3A to the nucleus. Incontrast, cells with PTEN deletion or FAK overexpression display a highlevel of phospho-FAK, phospho-AKT and cytoplasmic FOXO3A expression inboth the repertaxin treated and untreated cells. In all samples, nucleiwere counterstained with DAPI (in blue). C-D. The effect of Repertaxinon the SUM159 PTEN-siRNA and SUM159 Ad-FAK cell viability and on thecancer stem cell population was assessed utilizing the MTT and ALDEFLUORassays, respectively. After 3 days of treatment, cells with PTENdeletion or FAK overexpression developed resistance to repertaxin (C).Repertaxin treatment did not alter the proportion of ALDEFLUOR-positiveSUM159 PTEN knockdown cells. (D).

FIG. 22 shows the effect of repertaxin treatment on FAK/AKT activationin HCC1954 and MDA-MB-453 cell lines. To evaluate the effect ofrepertaxin treatment on CXCR1 downstream signaling we utilized alentiviral construct knocking down PTEN expression via a PTEN-siRNA A.HCC1954 control and HCC1954 PTEN-siRNA cells were cultured in adherentconditions for two days in the absence or presence of 100 nM repertaxinand the activation of the FAK/AKT pathway was accessed by westernblotting. Repertaxin treatment led to a decrease in FAK Tyr397 and AKTSer473 phosphorylation whereas PTEN deletion blocked the effect ofrepertaxin treatment on FAK and AKT activity. B. Repertaxin treatmentdid not have any effect on cell viability of MDA-MB-453 cell line whichharbor PTEN mutation. Utilizing western blot analysis we confirmed thatFAK/AKT pathway was not perturbated by repertaxin treatment.

FIG. 23 shows the effect of repertaxin on the HCC1954 PTEN-siRNA cellviability, assessed utilizing the MTT assay. After 3 days of treatment,cells with PTEN deletion developed resistance to repertaxin.

FIG. 24 shows expression of FAS-ligand and IL-8 mRNA after docetaxel orrepertaxin treatment measured by quantitative RT-PCR. A-B. SUM159 cellscultured in adherent condition were treated with repertaxin (100 nM),FAS agonist (500 ng/ml) or docetaxel (10 nM). After three days oftreatment cells were collected and RNA extracted. Docetaxel, inducedboth FAS-ligand (A) and IL-8 (B) mRNA in SUM159 cells. A 4-fold increaseof IL-8 mRNA level was detected after FAS agonist or docetaxel treatment(B).

FIG. 25 shows evaluation of PTEN/FAK/AKT activation in the threedifferent breast cancer xenografts. Western blot analysis revealed thatboth xenografts presented an expression of PTEN and an activation ofFAK/AKT pathway as shown by FAK Tyr397 and AKT Ser473 phosphorylation.

FIG. 26 shows Effect of Repertaxin treatment on the breast cancer stemcell population in vivo. A-C. To evaluate the effect of repertaxintreatment on tumor growth and the cancer stem cell population in vivo abreast cancer cell line (SUM159) and three human breast cancerxenografts generated from different patients (MC1, UM2, UM3) wereutilized. A. For each sample, 50,000 cells were injected into thehumanized mammary fat pad of NOD/SCID mice and monitored tumor size.When the tumors were about 4 mm, s.c. injection of repertaxin (15 mg/Kg)twice/day for 28 days or once/week I.P. injection of docetaxel (10mg/Kg) or the combination (repertaxin/docetaxel) was initiated. Thegraph shows the tumor size before and during the course of eachindicated treatment (arrow, beginning of the treatment). Similar resultswere observed for each sample with a statistically significant reductionof the tumor size in docetaxel alone or the combinationrepertaxin/docetaxel treated groups compared to the control, whereas nodifference was observed between the growth of the control tumors and thetumors treated with repertaxin alone. B-C. Evaluation of repertaxin,docetaxel, or the combined treatment on the cancer stem cell populationas assessed by the ALDEFLUOR assay (B) and by reimplantation intosecondary mice (C). Docetaxel-treated tumor xenografts showed similar orincrease percentage of ALDEFLUOR-positive cells compared to the control,whereas repertaxin treatment alone or in combination with docetaxelproduced a statistically significant decrease in ALDEFLUOR-positivecells with a 65% to 85% decrease in cancer stem cells compared to thecontrol (p<0.01) (B). Serial dilutions of cells obtained from primarytumors, non treated (control), and treated mice were implanted in themammary fat pad of secondary NOD/SCID mice which received no furthertreatment. Control and docetaxel treated primary tumors formed secondarytumors at all dilutions whereas, only higher numbers of cells obtainedfrom primary tumors treated with repertaxin or in combination withdocetaxel were able to form tumors. Furthermore, tumor growth wassignificantly delayed and resulting tumors were significantly smaller insize than the control or docetaxel treated tumors (C). D.Xenotransplants from each group were collected and immunohistochemistrystaining was done to detect the expression of phospho-FAK, phospho-AKT,FOXO3A, and ALDH1. Membranous phospho-FAK expression and cytoplasmicphospho-AKT expression (arrow) was detected in the control anddocetaxel-treated tumors whereas no expression was detected in thetumors treated with repertaxin alone or in combination with docetaxel.Nuclear FOXO3A expression (in brown) was detected in the cells treatedwith docetaxel or repertaxin alone or in combination. A decrease ofALDH1 expression (arrow) was detected in tumors treated with repertaxinalone or in combination compared to control and the docetaxel-treatedtumors.

FIG. 27 shows the effect of Repertaxin treatment on the breast cancerstem cell population in vivo. A-C. To evaluate the effect of repertaxintreatment on tumor growth and the cancer stem cell population in vivo, abreast cancer cell line (SUM159, A) and three human breast cancerxenografts generated from different patients. For each sample, 50,000cells were injected into the humanized mammary fat pad of NOD/SCID miceand monitored tumor size. When the tumors were about 4 mm, s.c.injection of repertaxin (15 mg/Kg) twice/day for 28 days or once/weekI.P. injection of docetaxel (10 mg/Kg) or the combination(repertaxin/docetaxel) was initiated. The graph shows the tumor sizebefore and during the course of each indicated treatment (arrow,beginning of the treatment). Similar results were observed for eachsample with a statistically significant reduction of the tumor size indocetaxel alone or the combination repertaxin/docetaxel treated groupscompared to the control whereas no difference was observed between thegrowth of the control tumors and the tumors treated with repertaxinalone. Evaluation of repertaxin, docetaxel, or the combined treatment onthe cancer stem cell population was assessed by the ALDEFLUOR assay andby reimplantation into secondary mice. Docetaxel-treated tumorxenografts showed similar or increased percentage of ALDEFLUOR-positivecells compared to the control, whereas repertaxin treatment alone or incombination with docetaxel produced a statistically significant decreasein ALDEFLUOR-positive cells with a 65% to 85% decrease in cancer stemcells compared to the control (p<0.01). Serial dilutions of cellsobtained from primary tumors, non-treated (control), and treated micewere implanted in the mammary fat pad of secondary NOD/SCID mice whichreceived no further treatment. Control and docetaxel treated primarytumors formed secondary tumors at all dilutions whereas, only highernumbers of cells obtained from primary tumors treated with repertaxin orin combination with docetaxel were able to form tumors. Tumor growth wassignificantly delayed and resulting tumors were significantly smaller insize than the control or docetaxel treated tumors.

FIG. 28 shows the effect of repertaxin treatment on the breast cancerstem cell population as assessed by the CD44+/CD24− phenotype. A-B.Evaluation of repertaxin, docetaxel, or the combined treatment on thecancer stem cell population was assessed by the presence of CD44+/CD24−cells. In residual tumors treated with docetaxel alone, we consistentlyobserved either an unchanged or increased percent of CD44+/CD24− cellswhereas repertaxin treatment alone or in combination with docetaxelresulted in a reduction of the CD44+/CD24− cell population. A. Flowchart analysis for UM3 xenograft is presented. B. Similar results wereobserved for MC1, UM2, and UM3. Almost all of SUM159 cells areCD44+/CD24− under all treatment conditions.

FIG. 29 shows repertaxin treatment reduces the development of systemicmetastasis. To evaluate the effect of repertaxin treatment on metastasisformation HCC1954 (A), SUM159 (B), MDA-MB-453 (C) breast cancer celllines were infected with a lentivirus expressing luciferase andinoculated 250,000 luciferase infected cells into NOD/SCID mice viaintracardial injection. Mice were treated 12 hours after theintracardiac injection either with s.c. injection of saline solution ors.c. injection of repertaxin (15 mg/kg), twice a day during 28 days.Metastasis formation was monitored using bioluminescence imaging.Quantification of the normalized photon flux measured at weeklyintervals following inoculation revealed a statistically significantdecrease in metastasis formation in repertaxin compared to salinecontrols for mice inoculated with HCC1954 or SUM159 cells (A-B). Incontrast, repertaxin treatment did not have any effect on metastasisformation for the mice injected with MDA-MB-453 cells. (C). Histologicconfirmation, by H&E staining, of metastasis in bone, and soft tissueresulting from mice not treated by repertaxin (D).

FIG. 30 shows IL-8/CXCR1 signalling in cancer stem cells treated withchemotherapy alone or in combination with repertaxin. A. Representationof potential IL-8/CXCR1 cell signaling in cancer stem cells. CXCR1activation following IL-8 binding induces phosphorylation of the FocalAdhesion Kinase (FAK). Active FAK phosphorylates AKT and activates theWNT pathway, which regulates stem cell self renewal and FOXO3A thatregulates cell survival. Activation of FAK protects cancer stem cellsfrom a FAS-ligand/FAS mediated bystander effect by inhibiting FADD, adownstream effector of FAS signaling. In the presence of chemotherapy,only the bulk tumor cells are sensitive to the treatment and release ahigh level of IL-8 and FAS-ligand proteins during the apoptotic process.Breast cancer stem cells are stimulated via an IL-8 mediated bystandereffect and are resistant to the bystander killing effect mediated viaFAS-ligand. B. Repertaxin treatment blocks IL-8/CXCR1 signaling andinhibits breast cancer stem cell self-renewal and survival. Whenrepertaxin treatment is combined with chemotherapy the cancer stem cellsare sensitized to the bystander killing effect mediated by FAS-ligand.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the terms “anticancer agent,” “conventional anticanceragent,” or “cancer therapeutic drug” refer to any therapeutic agents(e.g., chemotherapeutic compounds and/or molecular therapeuticcompounds), radiation therapies, or surgical interventions, used in thetreatment of cancer (e.g., in mammals).

As used herein, the terms “drug” and “chemotherapeutic agent” refer topharmacologically active molecules that are used to diagnose, treat, orprevent diseases or pathological conditions in a physiological system(e.g., a subject, or in vivo, in vitro, or ex vivo cells, tissues, andorgans). Drugs act by altering the physiology of a living organism,tissue, cell, or in vitro system to which the drug has beenadministered. It is intended that the terms “drug” and “chemotherapeuticagent” encompass anti-hyperproliferative and antineoplastic compounds aswell as other biologically therapeutic compounds.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations.

As used herein, the term “administration” refers to the act of giving adrug, prodrug, antibody, or other agent, or therapeutic treatment to aphysiological system (e.g., a subject or in vivo, in vitro, or ex vivocells, tissues, and organs). Exemplary routes of administration to thehuman body can be through the eyes (ophthalmic), mouth (oral), skin(transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal),ear, by injection (e.g., intravenously, subcutaneously, intratumorally,intraperitoneally, etc.) and the like.

“Coadministration” refers to administration of more than one chemicalagent or therapeutic treatment (e.g., radiation therapy) to aphysiological system (e.g., a subject or in vivo, in vitro, or ex vivocells, tissues, and organs). “Coadministration” of the respectivechemical agents (e.g. IL8-CXCR1 signaling pathway antagonist andadditional chemotherapeutic) may be concurrent, or in any temporal orderor physical combination.

As used herein, the term “regression” refers to the return of a diseasedsubject, cell, tissue, or organ to a non-pathological, or lesspathological state as compared to basal nonpathogenic exemplary subject,cell, tissue, or organ. For example, regression of a tumor includes areduction of tumor mass as well as complete disappearance of a tumor ortumors.

As used herein the term, “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes and cell cultures. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreactions that occur within a natural environment.

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g.,with an immortal phenotype), primary cell cultures, finite cell lines(e.g., non-transformed cells), and any other cell population maintainedin vitro, including oocytes and embryos.

As used herein, the term “subject” or “patient” refers to organisms tobe treated by the methods of the present invention. Such organismsinclude, but are not limited to, humans and veterinary animals (dogs,cats, horses, pigs, cattle, sheep, goats, and the like). In the contextof the invention, the term “subject” or “patient” generally refers to anindividual who will receive or who has received treatment.

The term “diagnosed,” as used herein, refers to the recognition of adisease by its signs and symptoms or genetic analysis, pathologicalanalysis, histological analysis, and the like.

As used herein, the term “antisense” is used in reference to nucleicacid sequences (e.g., RNA, phosphorothioate DNA) that are complementaryto a specific RNA sequence (e.g., mRNA). Included within this definitionare natural or synthetic antisense RNA molecules, including moleculesthat regulate gene expression, such as small interfering RNAs or microRNAs. One type of antisense sequence that may be employed by the presentinvention is the type that are specific for CXCR1 mRNA.

The term “test compound” or “candidate compound” refers to any chemicalentity, pharmaceutical, drug, and the like, that can be used to treat orprevent a disease, illness, sickness, or disorder of bodily function, orotherwise alter the physiological or cellular status of a sample. Testcompounds comprise both known and potential therapeutic compounds. Atest compound can be determined to be therapeutic by using the screeningmethods of the present invention. A “known therapeutic compound” refersto a therapeutic compound that has been shown (e.g., through animaltrials or prior experience with administration to humans) to beeffective in such treatment or prevention. In preferred embodiments,“test compounds” are anticancer agents. In particularly preferredembodiments, “test compounds” are anticancer agents that induceapoptosis in cells.

As used herein, the term “antigen binding protein” refers to proteinswhich bind to a specific antigen. “Antigen binding proteins” include,but are not limited to, immunoglobulins, including polyclonal,monoclonal, chimeric, single chain, and humanized antibodies, Fabfragments, F(ab′)2 fragments, and Fab expression libraries. Variousprocedures known in the art are used for the production of polyclonalantibodies. For the production of antibodies, various host animals canbe immunized by injection with the peptide corresponding to the desiredepitope including, but not limited to, rabbits, mice, rats, sheep,goats, etc. In a preferred embodiment, the peptide is conjugated to animmunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin(BSA), or keyhole limpet hemocyanin (KLH)). Various adjuvants are usedto increase the immunological response, depending on the host species,including, but not limited to, Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacteriumparvum.

For preparation of monoclonal antibodies, any technique that providesfor the production of antibody molecules by continuous cell lines inculture may be used (See e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).These include, but are not limited to, the hybridoma techniqueoriginally developed by Köhler and Milstein (Köhler and Milstein,Nature, 256:495-497 (1975)), as well as the trioma technique, the humanB-cell hybridoma technique (See e.g., Kozbor et al., Immunol. Today,4:72 (1983)), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96 (1985)).

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778; herein incorporated byreference) can be adapted to produce specific single chain antibodies asdesired. An additional embodiment of the invention utilizes thetechniques known in the art for the construction of Fab expressionlibraries (Huse et al., Science, 246:1275-1281 (1989)) to allow rapidand easy identification of monoclonal Fab fragments with the desiredspecificity.

Antibody fragments that contain the idiotype (antigen binding region) ofthe antibody molecule can be generated by known techniques. For example,such fragments include, but are not limited to: the F(ab′)2 fragmentthat can be produced by pepsin digestion of an antibody molecule; theFab′ fragments that can be generated by reducing the disulfide bridgesof an F(ab′)2 fragment, and the Fab fragments that can be generated bytreating an antibody molecule with papain and a reducing agent.

Genes encoding antigen-binding proteins can be isolated by methods knownin the art. In the production of antibodies, screening for the desiredantibody can be accomplished by techniques known in the art (e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), Western Blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc.) etc.

As used herein, the term “modulate” refers to the activity of a compoundto affect (e.g., to promote or retard) an aspect of the cellularfunction including, but not limited to, cell growth, proliferation,invasion, angiogenesis, apoptosis, and the like.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of treating cancer byadministering an IL8-CXCR1 pathway inhibitor (e.g., an anti-CXCR1antibody or Repertaxin) alone or in combination with an additionalchemotherapeutic agent such that non-tumorigenic and tumorigenic cancercells in a subject are killed. The present invention also providescompositions and methods for treating and diagnosing the presence ofsolid tumor stem cells in a patient (e.g., based on the presence ofCXCR1 or FBXO21).

I. Tumorigenic Cancer Cells, ALDH, CXCR1, and CXCR1 Inhibition

The evolution of a normal cell into a fully transformed one requires thederegulation of multiple cellular processes (1, 2). According toclassical models of carcinogenesis, these events can occur in any cell.In contrast, the “cancer stem cell hypothesis” holds that thepreferential targets of oncogenic transformation are tissue stem orearly progenitor cells that have acquired self-renewal potential (3-6).These “tumor-initiating cells” or “cancer stem cells” (CSC), in turn,are characterized by their ability to undergo self-renewal, a processthat drives tumorigenesis and differentiation which contributes to tumorcellular heterogeneity. Recent evidence supporting the cancer stem cellhypothesis has been generated utilizing xenografts of primary humantumors. These studies have suggested that tumors are composed of acellular hierarchy driven by the cancer stem cell component. Inaddition, recent data suggest that immortalized cell lines derived fromboth murine and human tissues may also contain a cellular populationdisplaying stem cell properties. Most of these studies have been basedon in vitro properties including clonogenic potential, sphere formationand multi-lineage differentiation potential (7-10). More limited studiesutilizing functional transplantation of immortalized cell lines inxenografts have also suggested the existence of such a hierarchy. Thesestudies have generally utilized Hoechst dye exclusion to identify theso-called “side population” (SP) (7, 9, 11). In addition, cell surfacemarkers defined using primary tumor xenografts such as CD44 and CD133have also been utilized to identify similar populations in establishedcell lines (7, 8).

As described in the Examples below, the expression of the stem cellmarker Aldehyde dehydrogenase (ALDH) was studied in a series of 33 celllines derived from human breast cancers and non-transformed breastcells. ALDH is a detoxifying enzyme responsible for the oxidation ofintracellular aldehydes and is thought to play a role in stem celldifferentiation through metabolism of retinal to retinoic acid (12, 13).ALDH activity as assessed by the fluorescent ALDEFLUOR assay has beensuccessfully utilized to isolate cancer stem cells in multiple myelomaand acute myeloid leukemia (AML) as well as from brain tumors (14-16).It was recently demonstrated that ALDH activity can be utilized toisolate a subpopulation of cells that display stem cell properties fromnormal human breast tissue and breast carcinomas (17). TheALDEFLUOR-positive population isolated from reduction mammoplasty tissueis able to reconstitute ductal alveolar structures in mammary fat padsof humanized NOD/SCID mice. Furthermore, ALDEFLUOR-positive cellsisolated from human mammary carcinomas have stem cell properties asdemonstrated by their ability to reconstitute tumors on serial passagein NOD/SCID mice as well as to generate the phenotypic heterogeneity ofthe initial tumors (17). In the Examples below, it is demonstrated thatthe majority of breast cancer cell lines contain an ALDEFLUOR-positivepopulation with a distinct molecular profile that displays cancer stemcell properties.

As described in the Examples below, work conducted during thedevelopment of embodiments of the present invention identified CXCR1(which is a receptor for the inflammatory chemokine IL8) as a cancerstem cell marker. Only cells within the Aldefluor-positive populationexpressed CXCR1. Furthermore, it was demonstrated that this receptorplays a functional role in that recombinant IL8 is able to increase thestem cell proportion in cell lines as determined by Aldefluor and sphereformation assays. Although IL8 has been reported to be associated withaggressive breast cancers and is higher in the serum women withmetastatic disease, it is believe that the present invention is thefirst to show a functional link between IL8 and its receptor CXCR1 instem cells.

As further described in the Examples below, it was demonstrated that onecan selectively target cancer stem cells by blocking the CXCR1 receptorin these cells. In one approach described in the Examples, breast cancercells lines were treated with monoclonal antibodies to CXCR1, but not tothe other IL8 receptor CXCR2. Such treatment selectively targeted cancerstem cells as demonstrated by reduced Aldefluor-positive populations.Remarkably, it found that although CXCR1 is only expressed in a verysmall percentage of cells (e.g., less than 1%), that blockade of theCXCR1 receptor induced cell death in the majority of other cancer cellsdespite the fact that they lack the CXCR1 receptor. The molecularpathway which mediates the effects of IL on cancer stem cells andaccounts for this so-called “bystander effect” of killing other cellshas been elucidated. IL8 stimulates stem cell self-renewal by binding toCXCR1, which in turn activates the focal adhesion kinase Fak pathway.This results in activation of Akt which drives stem cell self-renewal.When this pathway is blocked in cancer stem cells, the decrease in Aktsignaling causes cytoplasmic sequestration of the Foxo transcriptionfactors resulting in an increased synthesis of Fas ligand. Fas ligand issecreted from cancer stem cells and induces cell death in surroundingcells which contain the Fas receptor.

While the present invention is not limited to any particular mechanism,and an understanding of the mechanism is not necessary to practice thepresent invention, it is believed that CXCR1 mediates cancer stem cellself-renewal through a pathway involving Fak and Akt and that blockadeof this pathway induces cell death in cancer stem cells as well assurrounding tumor cells. As such, in certain embodiments, the presentinvention provides compositions and methods for disrupting the IL8-CXCR1pathway (e.g., with anti-CXCR1 antibodies, anti-FAK antibodies, or otheragents) in order to treat cancer.

Since IL8 is a chemokine involved in tissue inflammation, there has beenprevious interest in developing inhibitors of IL8 signaling. A smallmolecule inhibitor, Repartaxin, has been developed as ananti-inflammatory agent to potentially reduce complications ofmyocardial infarction and stroke. Repartaxin has been introduced intophase I and phase II clinical trials and has shown little toxicity. Asshown in the Examples below, Repartaxin (like anti-CXCR1 antibodies) isable to target cancer stem cells as well as to induce a Fas ligand fasmediated apoptosis by bystander effect in surrounding cells.Importantly, in tumor xenografts, Repartaxin potentiates the effect ofchemotherapy. Furthermore, unlike chemotherapy, which preferentiallydestroys the differentiated cells in tumors sparing the tumor stemcells, Repartaxin is able to target tumor stem cells. As shown in theexamples, this was demonstrated by a decrease in the Aldefluorpopulation in Repartaxin treated tumors and by the decrease in abilityof these treated tumor cells to form secondary tumors in mice. Alsotested was the effects of Repartaxin on the ability to block metastasis.Tumor cells were labeled with luciferase and injected intracardiac in anexperimental metastasis model. One day after the tumor cells wereintroduced, one group of animals was placed on repartaxin alone and theother no treatment. Repartaxin significantly reduced the development ofmetastasis.

The present invention identified the IL8 receptor CXCR1 as a target intreating cancer stem cells. The small molecule inhibitor Repartaxininhibits both CXCR1 and CXCR2. The Examples demonstrated that it isCXCR1 that is the most important receptor in cancer stem cells.Furthermore, the Examples indicate that the failure of cytotoxicchemotherapy to affectively treat established cancers may be not onlydue to the inability of this therapy to target cancer stem cells, but inaddition to the documented increase of IL8 secretion upon tumorcytotoxic chemotherapy treatment. The present Examples indicate that theuse of CXCR1 inhibitors have beneficial effects in being able tospecifically target cancer stem cells as well as to block the IL8stimulation of these cells induced by cytotoxic chemotherapy.

Targeting the IL8-CXCR1 pathway is not limited to breast cancer, butinstead, can be employed in any type of cancer. Preferably, the type ofcancer treated is one where there is evidence of increased IL8production (e.g., in conjunction with chemotherapy). Chemotherapy agentshave been shown to directly regulate IL8 transcription in cancer cells.Paclitaxel increases IL8 transcription and secretion in ovarian, breastand lung cancer cell lines (Uslu et al., 2005, Int. J. Gynecol. Cancer,15:240-245; and Collins et al., 2000, Can. Imm. Immuno., 49:78-84, bothof which are herein incorporated by reference). Also, administration ofadriamycin and 5-fluoro-2′-deoxyuridine to breast cancer cells (DeLarcoet al., 2001, Can. Res. 61:2857-2861, herein incorporated by reference),the addition of 5-FU to oral cancer cells (Tamatani et al., 2004, Int.,J. Can., 108:912:921, herein incorporated by reference), doxorubicinaddition to small cell lung cancer cells (Shibakura et al., 2003, Int.J. Can., 103:380-386, herein incorporated by reference) and dacarbazineadministration to melanoma cells (Lev et al., 2003, Mol., Can. Ther.,2:753-763, herein incorporated by reference) all result in increasedCXCL8 expression. As such, in certain embodiments, the present inventionprovides agents for targeting the IL-CXCR1, in combination with achemotherapy agents (e.g., such as those mentioned in the abovereferences) for treating a subject with a type of cancer including, butnot limited to, prostate cancer, ovarian cancer, breast cancer,melanoma, non-small cell lung cancer, small-cell lung cancer, andesophageal adenocarcinoma.

The present invention is not limited to the type of cancer treated andinstead includes, but is not limited to, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma.

II. Detection of Solid Tumor Stem Cell Cancer Markers

In some embodiments, the present invention provides methods fordetection of expression of stem cell cancer markers (e.g., CXCR1,FBXO21, NFYA, NOTCH2, RAD51L1, TBP, and other proteins from Table 1). Insome embodiments, expression is measured directly (e.g., at the RNA orprotein level). In some embodiments, expression is detected in tissuesamples (e.g., biopsy tissue). In other embodiments, expression isdetected in bodily fluids (e.g., including but not limited to, plasma,serum, whole blood, mucus, and urine).

The present invention further provides panels and kits for the detectionof markers. In some embodiments, the presence of a stem cell cancermarker is used to provide a prognosis to a subject. The informationprovided is also used to direct the course of treatment. For example, ifa subject is found to have a marker indicative of a solid tumor stemcell (e.g., CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1, TBP, and otherproteins from Table 1), additional therapies (e.g., radiation therapies)can be started at an earlier point when they are more likely to beeffective (e.g., before metastasis). In addition, if a subject is foundto have a tumor that is not responsive to certain therapy, the expenseand inconvenience of such therapies can be avoided.

In some embodiments, the present invention provides a panel for theanalysis of a plurality of markers (e.g., the combination of CXCR1 orFBXO21 and at least one of CD44, CD24, and ESA). The panel allows forthe simultaneous analysis of multiple markers correlating withcarcinogenesis and/or metastasis. Depending on the subject, panels canbe analyzed alone or in combination in order to provide the bestpossible diagnosis and prognosis. Markers for inclusion on a panel areselected by screening for their predictive value using any suitablemethod, including but not limited to, those described in theillustrative examples below.

1. Detection of RNA

In some embodiments, detection of solid tumor stem cell cancer markersare detected by measuring the expression of corresponding mRNA in atissue sample. mRNA expression can be measured by any suitable method,including but not limited to, those disclosed below. The accessionnumber for human CXCR1 nucleic acid is NM_000634 (herein incorporated byreference) and the accession number for human FBXO21 is NM_033624(herein incorporated by reference). These sequences can be used todesign primers and probes (as well as siRNA sequences).

In some embodiments, RNA is detected by Northern blot analysis. Northernblot analysis involves the separation of RNA and hybridization of acomplementary labeled probe.

In still further embodiments, RNA (or corresponding cDNA) is detected byhybridization to an oligonucleotide probe). A variety of hybridizationassays using a variety of technologies for hybridization and detectionare available. For example, in some embodiments, TaqMan assay (PEBiosystems, Foster City, Calif.; See e.g., U.S. Pat. Nos. 5,962,233 and5,538,848, each of which is herein incorporated by reference) isutilized. The assay is performed during a PCR reaction. The TaqMan assayexploits the 5′-3′ exonuclease activity of the AMPLITAQ GOLD DNApolymerase. A probe consisting of an oligonucleotide with a 5′-reporterdye (e.g., a fluorescent dye) and a 3′-quencher dye is included in thePCR reaction. During PCR, if the probe is bound to its target, the 5′-3′nucleolytic activity of the AMPLITAQ GOLD polymerase cleaves the probebetween the reporter and the quencher dye. The separation of thereporter dye from the quencher dye results in an increase offluorescence. The signal accumulates with each cycle of PCR and can bemonitored with a fluorimeter.

In yet other embodiments, reverse-transcriptase PCR (RT-PCR) is used todetect the expression of RNA. In RT-PCR, RNA is enzymatically convertedto complementary DNA or “cDNA” using a reverse transcriptase enzyme. ThecDNA is then used as a template for a PCR reaction. PCR products can bedetected by any suitable method, including but not limited to, gelelectrophoresis and staining with a DNA specific stain or hybridizationto a labeled probe. In some embodiments, the quantitative reversetranscriptase PCR with standardized mixtures of competitive templatesmethod described in U.S. Pat. Nos. 5,639,606, 5,643,765, and 5,876,978(each of which is herein incorporated by reference) is utilized.

2. Detection of Protein

In other embodiments, gene expression of stem cell cancer markers isdetected by measuring the expression of the corresponding protein orpolypeptide (e.g., CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1, TBP, and otherproteins from Table 1). Protein expression can be detected by anysuitable method. In some embodiments, proteins are detected byimmunohistochemistry. In other embodiments, proteins are detected bytheir binding to an antibody raised against the protein. The accessionnumber for human CXCR1 protein is NP_000625 (herein incorporated byreference) and the accession number for human FBXO21 is NP_296373(herein incorporated by reference). The generation of antibodies isdescribed below.

Antibody binding is detected by techniques known in the art (e.g.,radioimmunoassay, ELISA (enzyme linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (e.g., usingcolloidal gold, enzyme or radioisotope labels, for example), Westernblots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays, etc.), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many methods are known in the art for detecting binding in animmunoassay and are within the scope of the present invention.

In some embodiments, an automated detection assay is utilized. Methodsfor the automation of immunoassays include those described in U.S. Pat.Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which isherein incorporated by reference. In some embodiments, the analysis andpresentation of results is also automated. For example, in someembodiments, software that generates a prognosis based on the presenceor absence of a series of proteins corresponding to cancer markers isutilized.

In other embodiments, the immunoassay described in U.S. Pat. Nos.5,599,677 and 5,672,480; each of which is herein incorporated byreference.

3. cDNA Microarray Technology

cDNA microarrays are composed of multiple (usually thousands) ofdifferent cDNAs spotted (usually using a robotic spotting device) ontoknown locations on a solid support, such as a glass microscope slide.The cDNAs are typically obtained by PCR amplification of plasmid libraryinserts using primers complementary to the vector backbone portion ofthe plasmid or to the gene itself for genes where sequence is known. PCRproducts suitable for production of microarrays are typically between0.5 and 2.5 kB in length. Full length cDNAs, expressed sequence tags(ESTs), or randomly chosen cDNAs from any library of interest can bechosen. ESTs are partially sequenced cDNAs as described, for example, inHillier, et al., 1996, 6:807-828. Although some ESTs correspond to knowngenes, frequently very little or no information regarding any particularEST is available except for a small amount of 3′ and/or 5′ sequence and,possibly, the tissue of origin of the mRNA from which the EST wasderived. As will be appreciated by one of ordinary skill in the art, ingeneral the cDNAs contain sufficient sequence information to uniquelyidentify a gene within the human genome. Furthermore, in general thecDNAs are of sufficient length to hybridize, selectively, specificallyor uniquely, to cDNA obtained from mRNA derived from a single gene underthe hybridization conditions of the experiment.

In a typical microarray experiment, a microarray is hybridized withdifferentially labeled RNA, DNA, or cDNA populations derived from twodifferent samples. Most commonly RNA (either total RNA or poly A+RNA) isisolated from cells or tissues of interest and is reverse transcribed toyield cDNA. Labeling is usually performed during reverse transcriptionby incorporating a labeled nucleotide in the reaction mixture. Althoughvarious labels can be used, most commonly the nucleotide is conjugatedwith the fluorescent dyes Cy3 or Cy5. For example, Cy5-dUTP and Cy3-dUTPcan be used. cDNA derived from one sample (representing, for example, aparticular cell type, tissue type or growth condition) is labeled withone fluorophore while cDNA derived from a second sample (representing,for example, a different cell type, tissue type, or growth condition) islabeled with the second fluorophore. Similar amounts of labeled materialfrom the two samples are cohybridized to the microarray. In the case ofa microarray experiment in which the samples are labeled with Cy5 (whichfluoresces red) and Cy3 (which fluoresces green), the primary data(obtained by scanning the microarray using a detector capable ofquantitatively detecting fluorescence intensity) are ratios offluorescence intensity (red/green, R/G). These ratios represent therelative concentrations of cDNA molecules that hybridized to the cDNAsrepresented on the microarray and thus reflect the relative expressionlevels of the mRNA corresponding to each cDNA/gene represented on themicroarray.

Each microarray experiment can provide tens of thousands of data points,each representing the relative expression of a particular gene in thetwo samples. Appropriate organization and analysis of the data is of keyimportance, and various computer programs that incorporate standardstatistical tools have been developed to facilitate data analysis. Onebasis for organizing gene expression data is to group genes with similarexpression patterns together into clusters. A method for performinghierarchical cluster analysis and display of data derived frommicroarray experiments is described in Eisen et al., 1998, PNAS95:14863-14868. As described therein, clustering can be combined with agraphical representation of the primary data in which each data point isrepresented with a color that quantitatively and qualitativelyrepresents that data point. By converting the data from a large table ofnumbers into a visual format, this process facilitates an intuitiveanalysis of the data. Additional information and details regarding themathematical tools and/or the clustering approach itself can be found,for example, in Sokal & Sneath, Principles of numerical taxonomy, xvi,359, W. H. Freeman, San Francisco, 1963; Hartigan, Clusteringalgorithms, xiii, 351, Wiley, New York, 1975; Paull et al., 1989, J.Natl. Cancer Inst. 81:1088-92; Weinstein et al. 1992, Science258:447-51; van Osdol et al., 1994, J. Natl. Cancer Inst. 86:1853-9; andWeinstein et al., 1997, Science, 275:343-9.

Further details of the experimental methods used in the presentinvention are found in the Example below. Additional informationdescribing methods for fabricating and using microarrays is found inU.S. Pat. No. 5,807,522, which is herein incorporated by reference.Instructions for constructing microarray hardware (e.g., arrayers andscanners) using commercially available parts. Additional discussions ofmicroarray technology and protocols for preparing samples and performingmicroarray experiments are found in, for example, DNA arrays foranalysis of gene expression, Methods Enzymol, 303:179-205, 1999;Fluorescence-based expression monitoring using microarrays, MethodsEnzymol, 306: 3-18, 1999; and M. Schena (ed.), DNA Microarrays: APractical Approach, Oxford University Press, Oxford, UK, 1999.

4. Data Analysis

In some embodiments, a computer-based analysis program is used totranslate the raw data generated by the detection assay (e.g., thepresence, absence, or amount of a given marker or markers) into data ofpredictive value for a clinician. The clinician can access thepredictive data using any suitable means. Thus, in some embodiments, thepresent invention provides the further benefit that the clinician, whois not likely to be trained in genetics or molecular biology, need notunderstand the raw data. The data is presented directly to the clinicianin its most useful form. The clinician is then able to immediatelyutilize the information in order to optimize the care of the subject.

The present invention contemplates any method capable of receiving,processing, and transmitting the information to and from laboratoriesconducting the assays, information provides, medical personal, andsubjects. For example, in some embodiments of the present invention, asample (e.g., a biopsy or a serum or urine sample) is obtained from asubject and submitted to a profiling service (e.g., clinical lab at amedical facility, genomic profiling business, etc.), located in any partof the world (e.g., in a country different than the country where thesubject resides or where the information is ultimately used) to generateraw data. Where the sample comprises a tissue or other biologicalsample, the subject can visit a medical center to have the sampleobtained and sent to the profiling center, or subjects can collect thesample themselves and directly send it to a profiling center. Where thesample comprises previously determined biological information, theinformation can be directly sent to the profiling service by the subject(e.g., an information card containing the information can be scanned bya computer and the data transmitted to a computer of the profilingcenter using an electronic communication system). Once received by theprofiling service, the sample is processed and a profile is produced(e.g., expression data), specific for the diagnostic or prognosticinformation desired for the subject.

The profile data is then prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw expression data (e.g. examining a number of the markers),the prepared format can represent a diagnosis or risk assessment for thesubject, along with recommendations for particular treatment options.The data can be displayed to the clinician by any suitable method. Forexample, in some embodiments, the profiling service generates a reportthat can be printed for the clinician (e.g., at the point of care) ordisplayed to the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject can chose furtherintervention or counseling based on the results. In some embodiments,the data is used for research use. For example, the data can be used tofurther optimize the inclusion or elimination of markers as usefulindicators of a particular condition or stage of disease.

5. Kits

In yet other embodiments, the present invention provides kits for thedetection and characterization of cancer (e.g. for detecting one or moreof the markers, or for modulating the activity of a peptide expressed byone or more of markers). In some embodiments, the kits containantibodies specific for a cancer marker, in addition to detectionreagents and buffers. In other embodiments, the kits contain reagentsspecific for the detection of mRNA or cDNA (e.g., oligonucleotide probesor primers). In some embodiments, the kits contain all of the componentsnecessary and/or sufficient to perform a detection assay, including allcontrols, directions for performing assays, and any necessary softwarefor analysis and presentation of results.

Another embodiment of the present invention comprises a kit to test forthe presence of the polynucleotides or proteins. The kit can comprise,for example, an antibody for detection of a polypeptide or a probe fordetection of a polynucleotide. In addition, the kit can comprise areference or control sample; instructions for processing samples,performing the test and interpreting the results; and buffers and otherreagents necessary for performing the test. In other embodiments the kitcomprises pairs of primers for detecting expression of one or more ofthe genes of the solid tumor stem cell gene signature. In otherembodiments the kit comprises a cDNA or oligonucleotide array fordetecting expression of one or more of the genes of the solid tumor stemcell gene signature.

6. In Vivo Imaging

In some embodiments, in vivo imaging techniques are used to visualizethe expression of cancer markers in an animal (e.g., a human ornon-human mammal). For example, in some embodiments, cancer marker mRNA(e.g., CXCR1 or FBXO21 mRNA) or protein (e.g., CXCR1 or FBXO21 protein)is labeled using a labeled antibody specific for the cancer marker. Aspecifically bound and labeled antibody can be detected in an individualusing an in vivo imaging method, including, but not limited to,radionuclide imaging, positron emission tomography, computerized axialtomography, X-ray or magnetic resonance imaging method, fluorescencedetection, and chemiluminescent detection. Methods for generatingantibodies to the cancer markers of the present invention are describedbelow.

The in vivo imaging methods of the present invention are useful in thediagnosis of cancers that express the solid tumor stem cell cancermarkers of the present invention. In vivo imaging is used to visualizethe presence of a marker indicative of the cancer. Such techniques allowfor diagnosis without the use of an unpleasant biopsy. The in vivoimaging methods of the present invention are also useful for providingprognoses to cancer patients. For example, the presence of a markerindicative of cancer stem cells can be detected. The in vivo imagingmethods of the present invention can further be used to detectmetastatic cancers in other parts of the body.

In some embodiments, reagents (e.g., antibodies) specific for CXCR1 orFBXO21 are fluorescently labeled. The labeled antibodies are introducedinto a subject (e.g., orally or parenterally). Fluorescently labeledantibodies are detected using any suitable method (e.g., using theapparatus described in U.S. Pat. No. 6,198,107, herein incorporated byreference).

In other embodiments, antibodies are radioactively labeled. The use ofantibodies for in vivo diagnosis is well known in the art. Sumerdon etal., (Nucl. Med. Biol 17:247-254 [1990] have described an optimizedantibody-chelator for the radioimmunoscintographic imaging of tumorsusing Indium-111 as the label. Griffin et al., (J Clin Onc 9:631-640[1991]) have described the use of this agent in detecting tumors inpatients suspected of having pancreatic cancer. The use of similaragents with paramagnetic ions as labels for magnetic resonance imagingis known in the art (Lauffer, Magnetic Resonance in Medicine 22:339-342[1991]). The label used will depend on the imaging modality chosen.Radioactive labels such as Indium-111, Technetium-99m, or Iodine-131 canbe used for planar scans or single photon emission computed tomography(SPECT). Positron emitting labels such as Fluorine-19 can also be usedfor positron emission tomography (PET). For MRI, paramagnetic ions suchas Gadolinium (III) or Manganese (II) can be used.

Radioactive metals with half-lives ranging from 1 hour to 3.5 days areavailable for conjugation to antibodies, such as scandium-47 (3.5 days)gallium-67 (2.8 days), gallium-68 (68 minutes), technetium-99m (6hours), and indium-111 (3.2 days), of which gallium-67, technetium-99m,and indium-111 are preferable for gamma camera imaging, gallium-68 ispreferable for positron emission tomography.

A useful method of labeling antibodies with such radiometals is by meansof a bifunctional chelating agent, such as diethylenetriaminepentaaceticacid (DTPA), as described, for example, by Khaw et al. (Science 209:295[1980]) for In-111 and Tc-99m, and by Scheinberg et al. (Science215:1511 [1982]). Other chelating agents can also be used, but the1-(p-carboxymethoxybenzyl)EDTA and the carboxycarbonic anhydride of DTPAare advantageous because their use permits conjugation without affectingthe antibody's immunoreactivity substantially.

Another method for coupling DPTA to proteins is by use of the cyclicanhydride of DTPA, as described by Hnatowich et al. (Int. J. Appl.Radiat. Isot. 33:327 [1982]) for labeling of albumin with In-111, butwhich can be adapted for labeling of antibodies. A suitable method oflabeling antibodies with Tc-99m which does not use chelation with DPTAis the pretinning method of Crockford et al., (U.S. Pat. No. 4,323,546,herein incorporated by reference).

A method of labeling immunoglobulins with Tc-99m is that described byWong et al. (Int. J. Appl. Radiat. Isot., 29:251 [1978]) for plasmaprotein, and recently applied successfully by Wong et al. (J. Nucl.Med., 23:229 [1981]) for labeling antibodies.

In the case of the radiometals conjugated to the specific antibody, itis likewise desirable to introduce as high a proportion of theradiolabel as possible into the antibody molecule without destroying itsimmunospecificity. A further improvement can be achieved by effectingradiolabeling in the presence of the specific stem cell cancer marker ofthe present invention, to insure that the antigen binding site on theantibody will be protected.

In still further embodiments, in vivo biophotonic imaging (Xenogen,Almeda, Calif.) is utilized for in vivo imaging. This real-time in vivoimaging utilizes luciferase. The luciferase gene is incorporated intocells, microorganisms, and animals (e.g., as a fusion protein with acancer marker of the present invention). When active, it leads to areaction that emits light. A CCD camera and software is used to capturethe image and analyze it.

III. Antibodies and Antibody Fragments

The present invention provides isolated antibodies and antibodyfragments against CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1, TBP, and otherproteins from Table 1. The antibody, or antibody fragment, can be anymonoclonal or polyclonal antibody that specifically recognizes theseproteins. In some embodiments, the present invention provides monoclonalantibodies, or fragments thereof, that specifically bind to CXCR1,FBXO21, NFYA, NOTCH2, RAD51L1, TBP, and other proteins from Table 1. Insome embodiments, the monoclonal antibodies, or fragments thereof, arechimeric or humanized antibodies that specifically bind to theseproteins. In other embodiments, the monoclonal antibodies, or fragmentsthereof, are human antibodies that specifically bind to these proteins.

The antibodies against CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1, TBP, andother proteins from Table 1 find use in the experimental, diagnostic andtherapeutic methods described herein. In certain embodiments, theantibodies of the present invention are used to detect the expression ofa cancer stem cell marker protein in biological samples such as, forexample, a patient tissue biopsy, pleural effusion, or blood sample.Tissue biopsies can be sectioned and protein detected using, forexample, immunofluorescence or immunohistochemistry. Alternatively,individual cells from a sample are isolated, and protein expressiondetected on fixed or live cells by FACS analysis. Furthermore, theantibodies can be used on protein arrays to detect expression of acancer stem cell marker, for example, on tumor cells, in cell lysates,or in other protein samples. In other embodiments, the antibodies of thepresent invention are used to inhibit the growth of tumor cells bycontacting the antibodies with tumor cells either in vitro cell basedassays or in vivo animal models. In still other embodiments, theantibodies are used to treat cancer in a human patient by administeringa therapeutically effective amount of an antibody against a cancer stemcell marker (e.g., from Table 1).

Polyclonal antibodies can be prepared by any known method. Polyclonalantibodies can be raised by immunizing an animal (e.g. a rabbit, rat,mouse, donkey, etc) by multiple subcutaneous or intraperitonealinjections of the relevant antigen (a purified peptide fragment,full-length recombinant protein, fusion protein, etc) optionallyconjugated to keyhole limpet hemocyanin (KLH), serum albumin, etc.diluted in sterile saline and combined with an adjuvant (e.g. Completeor Incomplete Freund's Adjuvant) to form a stable emulsion. Thepolyclonal antibody is then recovered from blood, ascites and the like,of an animal so immunized. Collected blood is clotted, and the serumdecanted, clarified by centrifugation, and assayed for antibody titer.The polyclonal antibodies can be purified from serum or ascitesaccording to standard methods in the art including affinitychromatography, ion-exchange chromatography, gel electrophoresis,dialysis, etc.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized as described above to elicit the production by lymphocytes ofantibodies that will specifically bind to an immunizing antigen.Alternatively, lymphocytes can be immunized in vitro. Followingimmunization, the lymphocytes are isolated and fused with a suitablemyeloma cell line using, for example, polyethylene glycol, to formhybridoma cells that can then be selected away from unfused lymphocytesand myeloma cells. Hybridomas that produce monoclonal antibodiesdirected specifically against a chosen antigen as determined byimmunoprecipitation, immunoblotting, or by an in vitro binding assaysuch as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay(ELISA) can then be propagated either in vitro culture using standardmethods (Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, 1986) or in vivo as ascites tumors in an animal. Themonoclonal antibodies can then be purified from the culture medium orascites fluid as described for polyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated, such as from mature B-cellsor hybridoma cell, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries as described (McCafferty et al., 1990, Nature,348:552-554; Clackson et al., 1991, Nature, 352:624-628; and Marks etal., 1991, J. Mol. Biol., 222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In one embodiment, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In otherembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Furthermore,site-directed or high-density mutagenesis of the variable region can beused to optimize specificity, affinity, etc. of a monoclonal antibody.

In some embodiments, of the present invention the monoclonal antibodyagainst a cancer stem cell marker is a humanized antibody. Humanizedantibodies are antibodies that contain minimal sequences from non-human(e.g., murine) antibodies within the variable regions. Such antibodiesare used therapeutically to reduce antigenicity and HAMA (humananti-mouse antibody) responses when administered to a human subject. Inpractice, humanized antibodies are typically human antibodies withminimum to no non-human sequences. A human antibody is an antibodyproduced by a human or an antibody having an amino acid sequencecorresponding to an antibody produced by a human.

Humanized antibodies can be produced using various techniques known inthe art. An antibody can be humanized by substituting the CDR of a humanantibody with that of a non-human antibody (e.g. mouse, rat, rabbit,hamster, etc.) having the desired specificity, affinity, and capability(Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988,Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536).The humanized antibody can be further modified by the substitution ofadditional residue either in the Fv framework region and/or within thereplaced non-human residues to refine and optimize antibody specificity,affinity, and/or capability.

Human antibodies can be directly prepared using various techniques knownin the art. Immortalized human B lymphocytes immunized in vitro orisolated from an immunized individual that produce an antibody directedagainst a target antigen can be generated (See, for example, Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); Boemer et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat.No. 5,750,373). Also, the human antibody can be selected from a phagelibrary, where that phage library expresses human antibodies (Vaughan etal., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS,95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Markset al., 1991, J. Mol. Biol., 222:581). Humanized antibodies can also bemade in transgenic mice containing human immunoglobulin loci that arecapable upon immunization of producing the full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Thisapproach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;5,625,126; 5,633,425; and 5,661,016.

This invention also encompasses bispecific antibodies that specificallyrecognize cancer stem cell markers. Bispecific antibodies are antibodiesthat are capable of specifically recognizing and binding at least twodifferent epitopes.

Bispecific antibodies can be intact antibodies or antibody fragments.Techniques for making bispecific antibodies are common in the art(Millstein et al., 1983, Nature 305:537-539; Brennan et al., 1985,Science 229:81; Suresh et al, 1986, Methods in Enzymol. 121:120;Traunecker et al., 1991, EMBO J. 10:3655-3659; Shalaby et al., 1992, J.Exp. Med. 175:217-225; Kostelny et al., 1992, J. Immunol. 148:1547-1553;Gruber et al., 1994, J. Immunol. 152:5368; and U.S. Pat. No. 5,731,168).

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. Various techniques are known for theproduction of antibody fragments. Traditionally, these fragments arederived via proteolytic digestion of intact antibodies (for exampleMorimoto et al., 1993, Journal of Biochemical and Biophysical Methods24:107-117 and Brennan et al., 1985, Science, 229:81). However, thesefragments are now typically produced directly by recombinant host cellsas described above. Thus Fab, Fv, and scFv antibody fragments can all beexpressed in and secreted from E. coli or other host cells, thusallowing the production of large amounts of these fragments.Alternatively, such antibody fragments can be isolated from the antibodyphage libraries discussed above. The antibody fragment can also belinear antibodies as described in U.S. Pat. No. 5,641,870, for example,and can be monospecific or bispecific. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner.

It may further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent. Cytotoxic agents includechemotherapeutic agents, growth inhibitory agents, toxins (e.g., anenzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), radioactive isotopes (i.e., aradioconjugate), etc. Chemotherapeutic agents useful in the generationof such immunoconjugates include, for example, methotrexate, adriamicin,doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents. Enzymatically active toxins and fragments thereofthat can be used include diphtheria A chain, nonbinding active fragmentsof diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, andthe tricothecenes. A variety of radionuclides are available for theproduction of radioconjugated antibodies including 212Bi, 131I, 131In,90Y, and 186Re. Conjugates of the antibody and cytotoxic agent are madeusing a variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Conjugates of an antibody and one ormore small molecule toxins, such as a calicheamicin, maytansinoids, atrichothene, and CC 1065, and the derivatives of these toxins that havetoxin activity, can also be used.

In some embodiments the antibody of the invention contains human Fcregions that are modified to enhance effector function, for example,antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complementdependent cytotoxicity (CDC). This can be achieved by introducing one ormore amino acid substitutions in an Fc region of the antibody. Forexample, cysteine residue(s) can be introduced in the Fc region to allowinterchain disulfide bond formation in this region to improvecomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC) (Caron et al., 1992, J. Exp Med. 176:1191-1195;Shopes, 1992, Immunol. 148:2918-2922). Homodimeric antibodies withenhanced anti-tumor activity can also be prepared usingheterobifunctional cross-linkers as described in Wolff et al., 1993,Cancer Research 53:2560-2565. Alternatively, an antibody can beengineered which has dual Fc regions (Stevenson et al., 1989,Anti-Cancer Drug Design 3:219-230).

IV. Drug Screening

In some embodiments, the present invention provides drug screeningassays (e.g., to screen for anticancer drugs). The screening methods ofthe present invention utilize stem cell cancer markers (e.g., CXCR1,FBXO21, NFYA, NOTCH2, RAD51L1, TBP, and other proteins from Table 1)identified using the methods of the present invention. For example, insome embodiments, the present invention provides methods of screeningfor compounds that alter (e.g., increase or decrease) the expression of,or activity of, CXCR1 or FBXO21. In some embodiments, candidatecompounds are antisense agents or siRNA agents (e.g., oligonucleotides)directed against cancer markers. In other embodiments, candidatecompounds are antibodies that specifically bind to a stem cell cancermarker of the present invention. In certain embodiments, libraries ofcompounds of small molecules are screened using the methods describedherein.

In one screening method, candidate compounds are evaluated for theirability to alter stem cell cancer marker expression by contacting acompound with a cell expressing a stem cell cancer marker and thenassaying for the effect of the candidate compounds on expression. Insome embodiments, the effect of candidate compounds on expression of acancer marker gene is assayed by detecting the level of cancer markermRNA expressed by the cell. mRNA expression can be detected by anysuitable method. In other embodiments, the effect of candidate compoundson expression of cancer marker genes is assayed by measuring the levelof polypeptide encoded by the cancer markers. The level of polypeptideexpressed can be measured using any suitable method, including but notlimited to, those disclosed herein. In some embodiments, other changesin cell biology (e.g., apoptosis) are detected.

Specifically, the present invention provides screening methods foridentifying modulators, i.e., candidate or test compounds or agents(e.g., proteins, peptides, peptidomimetics, peptoids, small molecules orother drugs) which bind to, or alter the signaling or functionassociated with the cancer markers of the present invention, have aninhibitory (or stimulatory) effect on, for example, stem cell cancermarker expression or cancer markers activity, or have a stimulatory orinhibitory effect on, for example, the expression or activity of acancer marker substrate. Compounds thus identified can be used tomodulate the activity of target gene products (e.g., stem cell cancermarker genes, such as CXCR1 or FBXO21) either directly or indirectly ina therapeutic protocol, to elaborate the biological function of thetarget gene product, or to identify compounds that disrupt normal targetgene interactions. Compounds which inhibit the activity or expression ofcancer markers are useful in the treatment of proliferative disorders,e.g., cancer, particularly metastatic cancer or eliminating orcontrolling tumor stem cells to prevent or reduce the risk of cancer.

In one embodiment, the invention provides assays for screening candidateor test compounds that are substrates of a cancer markers protein orpolypeptide or a biologically active portion thereof. In anotherembodiment, the invention provides assays for screening candidate ortest compounds that bind to or modulate the activity of a cancer markerprotein or polypeptide or a biologically active portion thereof.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone, which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckennann et al., J.Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are preferred for use withpeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90:6909 [1993]; Erb et al., Proc. Nad. Acad. Sci. USA 91:11422[1994]; Zuckermann et al., J. Med. Chem. 37:2678 [1994]; Cho et al.,Science 261:1303 [1993]; Carrell et al., Angew. Chem. Int. Ed. Engl.33.2059 [1994]; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061[1994]; and Gallop et al., J. Med. Chem. 37:1233 [1994].

Libraries of compounds can be presented in solution (e.g., Houghten,Biotechniques 13:412-421 [1992]), or on beads (Lam, Nature 354:82-84[1991]), chips (Fodor, Nature 364:555-556 [1993]), bacteria or spores(U.S. Pat. No. 5,223,409; herein incorporated by reference), plasmids(Cull et al., Proc. Nad. Acad. Sci. USA 89:18651869 [1992]) or on phage(Scott and Smith, Science 249:386-390 [1990]; Devlin Science 249:404-406[1990]; Cwirla et al., Proc. Natl. Acad. Sci. 87:6378-6382 [1990];Felici, J. Mol. Biol. 222:301 [1991]).

In one embodiment, an assay is a cell-based assay in which a cell thatexpresses a stem cell cancer marker protein or biologically activeportion thereof is contacted with a test compound, and the ability ofthe test compound to the modulate cancer marker's activity isdetermined. Determining the ability of the test compound to modulatestem cell cancer marker activity can be accomplished by monitoring, forexample, changes in enzymatic activity. The cell, for example, can be ofmammalian origin.

The ability of the test compound to modulate cancer marker binding to acompound, e.g., a stem cell cancer marker substrate, can also beevaluated. This can be accomplished, for example, by coupling thecompound, e.g., the substrate, with a radioisotope or enzymatic labelsuch that binding of the compound, e.g., the substrate, to a cancermarker can be determined by detecting the labeled compound, e.g.,substrate, in a complex.

Alternatively, the stem cell cancer marker is coupled with aradioisotope or enzymatic label to monitor the ability of a testcompound to modulate cancer marker binding to a cancer markers substratein a complex. For example, compounds (e.g., substrates) can be labeledwith 125I, 35S 14C or 3H, either directly or indirectly, and theradioisotope detected by direct counting of radioemmission or byscintillation counting. Alternatively, compounds can be enzymaticallylabeled with, for example, horseradish peroxidase, alkaline phosphatase,or luciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product.

The ability of a compound (e.g., a stem cell cancer marker substrate) tointeract with a stem cell cancer marker with or without the labeling ofany of the interactants can be evaluated. For example, amicrophysiometer can be used to detect the interaction of a compoundwith a cancer marker without the labeling of either the compound or thecancer marker (McConnell et al. Science 257:1906-1912 [1992]). As usedherein, a “microphysiometer” (e.g., Cytosensor) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a compound and cancer markers.

In yet another embodiment, a cell-free assay is provided in which acancer marker protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tobind to the stem cell cancer marker protein or biologically activeportion thereof is evaluated. Biologically active portions of the cancermarkers proteins to be used in assays of the present invention includefragments that participate in interactions with substrates or otherproteins, e.g., fragments with high surface probability scores.

Cell-free assays involve preparing a reaction mixture of the target geneprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FRET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No.4,968,103; each of which is herein incorporated by reference). Afluorophore label is selected such that a first donor molecule's emittedfluorescent energy will be absorbed by a fluorescent label on a second,‘acceptor’ molecule, which in turn is able to fluoresce due to theabsorbed energy.

Alternately, the ‘donor’ protein molecule can simply utilize the naturalfluorescent energy of tryptophan residues. Labels are chosen that emitdifferent wavelengths of light, such that the ‘acceptor’ molecule labelcan be differentiated from that of the ‘donor’. Since the efficiency ofenergy transfer between the labels is related to the distance separatingthe molecules, the spatial relationship between the molecules can beassessed. In a situation in which binding occurs between the molecules,the fluorescent emission of the ‘acceptor’ molecule label in 1 5 theassay should be maximal. An FRET binding event can be convenientlymeasured through standard fluorometric detection means well known in theart (e.g., using a fluorimeter).

In another embodiment, determining the ability of the stem cell cancermarkers protein to bind to a target molecule can be accomplished usingreal-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolanderand Urbaniczky, Anal. Chem. 63:2338-2345 [1991] and Szabo et al. Curr.Opin. Struct. Biol. 5:699-705 [1995]). “Surface plasmon resonance” or“BIA” detects biospecific interactions in real time, without labelingany of the interactants (e.g., BIAcore). Changes in the mass at thebinding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance (SPR)), resulting in a detectable signalthat can be used as an indication of real-time reactions betweenbiological molecules.

In one embodiment, the target gene product or the test substance isanchored onto a solid phase. The target gene product/test compoundcomplexes anchored on the solid phase can be detected at the end of thereaction. The target gene product can be anchored onto a solid surface,and the test compound, (which is not anchored), can be labeled, eitherdirectly or indirectly, with detectable labels discussed herein.

It may be desirable to immobilize stem cell cancer markers, ananti-cancer marker antibody or its target molecule to facilitateseparation of complexed from non-complexed forms of one or both of theproteins, as well as to accommodate automation of the assay. Binding ofa test compound to a stem cell cancer marker protein, or interaction ofa cancer marker protein with a target molecule in the presence andabsence of a candidate compound, can be accomplished in any vesselsuitable for containing the reactants. Examples of such vessels includemicrotiter plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows one or both of the proteins to be bound to a matrix. For example,glutathione-S-transferase-cancer marker fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione Sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione-derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or cancer marker protein, and the mixture incubated underconditions conducive for complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above.

Alternatively, the complexes can be dissociated from the matrix, and thelevel of cancer markers binding or activity determined using standardtechniques. Other techniques for immobilizing either cancer markersprotein or a target molecule on matrices include using conjugation ofbiotin and streptavidin. Biotinylated cancer marker protein or targetmolecules can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with,e.g., a labeled anti-IgG antibody).

This assay is performed utilizing antibodies reactive with stem cellcancer marker protein or target molecules but which do not interferewith binding of the stem cell cancer markers protein to its targetmolecule. Such antibodies can be derivatized to the wells of the plate,and unbound target or cancer markers protein trapped in the wells byantibody conjugation. Methods for detecting such complexes, in additionto those described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the cancermarker protein or target molecule, as well as enzyme-linked assays whichrely on detecting an enzymatic activity associated with the cancermarker protein or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including, butnot limited to: differential centrifugation (see, for example, Rivas andMinton, Trends Biochem Sci 18:284-7 [1993]); chromatography (gelfiltration chromatography, ion-exchange chromatography); electrophoresis(see, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology1999, J. Wiley: New York.); and immunoprecipitation (see, for example,Ausubel et al., eds. Current Protocols in Molecular Biology 1999, J.Wiley: New York). Such resins and chromatographic techniques are knownto one skilled in the art (See e.g., Heegaard J. Mol. Recognit 11:141-8[1998]; Hageand Tweed J. Chromatogr. Biomed. Sci. Appl 699:499-525[1997]). Further, fluorescence energy transfer can also be convenientlyutilized, as described herein, to detect binding without furtherpurification of the complex from solution.

The assay can include contacting the stem cell cancer markers protein orbiologically active portion thereof with a known compound that binds thecancer marker to form an assay mixture, contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with a cancer marker protein, wherein determining theability of the test compound to interact with a cancer marker proteinincludes determining the ability of the test compound to preferentiallybind to cancer markers or biologically active portion thereof, or tomodulate the activity of a target molecule, as compared to the knowncompound.

To the extent that stem cell cancer markers can, in vivo, interact withone or more cellular or extracellular macromolecules, such as proteins,inhibitors of such an interaction are useful. A homogeneous assay can beused can be used to identify inhibitors.

For example, a preformed complex of the target gene product and theinteractive cellular or extracellular binding partner product isprepared such that either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496, hereinincorporated by reference, that utilizes this approach forimmunoassays). The addition of a test substance that competes with anddisplaces one of the species from the preformed complex will result inthe generation of a signal above background. In this way, testsubstances that disrupt target gene product-binding partner interactioncan be identified. Alternatively, cancer markers protein can be used asa “bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al., Cell 72:223-232 [1993]; Maduraet al., J. Biol. Chem. 268.12046-12054 [1993]; Bartel et al.,Biotechniques 14:920-924 [1993]; Iwabuchi et al., Oncogene 8:1693-1696[1993]; and Brent WO 94/10300; each of which is herein incorporated byreference), to identify other proteins, that bind to or interact withcancer markers (“cancer marker-binding proteins” or “cancer marker-bp”)and are involved in cancer marker activity. Such cancer marker-bps canbe activators or inhibitors of signals by the cancer marker proteins ortargets as, for example, downstream elements of a cancermarkers-mediated signaling pathway.

Modulators of cancer markers expression can also be identified. Forexample, a cell or cell free mixture is contacted with a candidatecompound and the expression of cancer marker mRNA or protein evaluatedrelative to the level of expression of stem cell cancer marker mRNA orprotein in the absence of the candidate compound. When expression ofcancer marker mRNA or protein is greater in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of cancer marker mRNA or protein expression.Alternatively, when expression of cancer marker mRNA or protein is less(i.e., statistically significantly less) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as an inhibitor of cancer marker mRNA or protein expression.The level of cancer markers mRNA or protein expression can be determinedby methods described herein for detecting cancer markers mRNA orprotein.

This invention further pertains to novel agents identified by theabove-described screening assays (See e.g., below description of cancertherapies). Accordingly, it is within the scope of this invention tofurther use an agent identified as described herein (e.g., a cancermarker modulating agent, an antisense cancer marker nucleic acidmolecule, a siRNA molecule, a cancer marker specific antibody, or acancer marker-binding partner) in an appropriate animal model (such asthose described herein) to determine the efficacy, toxicity, sideeffects, or mechanism of action, of treatment with such an agent.Furthermore, novel agents identified by the above-described screeningassays can be, e.g., used for treatments as described herein (e.g. totreat a human patient who has cancer).

In certain embodiments, the present invention employs non-adherentmammospheres for various screening procedures, including methods forscreening CXCR1 or FBXO21 signaling pathway antagonists. Non-adherentmammospheres are an in vitro culture system that allows for thepropagation of primary human mammary epithelial stem and progenitorcells in an undifferentiated state, based on their ability toproliferate in suspension as spherical structures. Non-adherentmammospheres have previously been described in Dontu et al Genes Dev.2003 May 15; 17(10):1253-70, and Dontu et al., Breast Cancer Res. 2004;6(6):R605-15, both of which are herein incorporated by reference. Thesereferences are incorporated by reference in their entireties andspecifically for teaching the construction and use of non-adherentmammospheres. As described in Dontu et al., mammospheres have beencharacterized as being composed of stem and progenitor cells capable ofself-renewal and multi-lineage differentiation. Dontu et al. alsodescribes that mammospheres contain cells capable of clonally generatingcomplex functional ductal-alveolar structures in reconstituted 3-Dculture systems in Matrigel.

In certain embodiments, the following exemplary screening methods areemployed. For in vitro studies, one could treat cells with eitheradenoviral constructs expressing control or CXCR1 or FBXO21 candidatesiRNA (m.o.i. 10 to 100) for 3 days or a small molecule candidate (e.g.,PHA665752 derivative) (0.1-0.5 uM) for 3 days and compare the ability ofCXCR1+ or FBXO21+ cells to form tumor spheres compared in untreated vs.treated cells. For in vivo studies, one could infect human breast cancercells with a lentivirus expressing luciferase to monitor tumor growth.Luciferase-expressing cancer cells could be injected into breast tissueand tumors of approximately 0.5-0.7 cm in size could be established,with 5 animals per group. Animals with established tumors could then betreated with either a candidate CXCR1 or FBXO21 inhibitor (daily i.v. 30mg/kg/day for 7 days), or vehicle control. Parallel studies could beperformed using infection with adenovirus expressing control orcandidate CXCR1 or FBXO21 siRNA (m.o.i. 100 or 500 for 7 days). Animalscould be imaged at day 7, 14, 21, and 28 to assess tumor size and thenbe sacrificed. Tumor size could be further assessed at autopsy and aportion of the tumor stained to assess tumor histology. The remainingtumor could be harvested and sorted to assess the percentage of CXCR1 orFBXO21 positive and CXCR1 or FBXO21 negative cells. To verify thatadministration of candidate CXCR1 or FBXO21 inhibitor and candidateCXCR1 or FBXO21 siRNA adenovirus infection is inhibiting CXCR1 or FBXO21signaling function, phosphorylation of downstream mediators such asGab-1 and ERK could be examined (see, Chistensen et al., Cancer Res.,2003; 63:7345-7355, herein incorporated by reference).

V. Cancer Therapies

In some embodiments, the present invention provides therapies forcancer. In some embodiments, therapies target cancer markers (e.g.,including but not limited to, CXCR1 or FBXO21 and proteins in the CXCR1or FBXO21 signaling pathway). In some embodiments, any known or laterdeveloped cancer stem cell therapy may be used. For example, cancer stemcell therapeutic agents are described in U.S. Pat. Nos. 6,984,522 and7,115,360 and applications WO03/050502, WO05/074633, and WO05/005601,herein incorporated by reference in their entireties.

Antibody Therapy

In some embodiments, the present invention provides antibodies thattarget tumors that express a stem cell cancer marker of the presentinvention. Any suitable antibody (e.g., monoclonal, polyclonal, orsynthetic) can be utilized in the therapeutic methods disclosed herein.In some embodiments, the antibodies used for cancer therapy arehumanized antibodies. Methods for humanizing antibodies are well knownin the art (See e.g., U.S. Pat. Nos. 6,180,370, 5,585,089, 6,054,297,and 5,565,332; each of which is herein incorporated by reference).

In some embodiments, the therapeutic antibodies comprise an antibodygenerated against a stem cell cancer marker of the present invention,wherein the antibody is conjugated to a cytotoxic agent. In suchembodiments, a tumor specific therapeutic agent is generated that doesnot target normal cells, thus reducing many of the detrimental sideeffects of traditional chemotherapy. For certain applications, it isenvisioned that the therapeutic agents will be pharmacologic agents thatwill serve as useful agents for attachment to antibodies, particularlycytotoxic or otherwise anticellular agents having the ability to kill orsuppress the growth or cell division of endothelial cells. The presentinvention contemplates the use of any pharmacologic agent that can beconjugated to an antibody, and delivered in active form. Exemplaryanticellular agents include chemotherapeutic agents, radioisotopes, andcytotoxins. The therapeutic antibodies of the present invention caninclude a variety of cytotoxic moieties, including but not limited to,radioactive isotopes (e.g., iodine-131, iodine-123, technetium-99m,indium-111, rhenium-188, rhenium-186, gallium-67, copper-67, yttrium-90,iodine-125 or astatine-211), hormones such as a steroid, antimetabolitessuch as cytosines (e.g., arabinoside, fluorouracil, methotrexate oraminopterin; an anthracycline; mitomycin C), vinca alkaloids (e.g.,demecolcine; etoposide; mithramycin), and antitumor alkylating agentsuch as chlorambucil or melphalan. Other embodiments can include agentssuch as a coagulant, a cytokine, growth factor, bacterial endotoxin orthe lipid A moiety of bacterial endotoxin. For example, in someembodiments, therapeutic agents will include plant-, fungus- orbacteria-derived toxin, such as an A chain toxins, a ribosomeinactivating protein, α-sarcin, aspergillin, restrictocin, aribonuclease, diphtheria toxin or pseudomonas exotoxin, to mention justa few examples. In some embodiments, deglycosylated ricin A chain isutilized.

In any event, it is proposed that agents such as these can, if desired,be successfully conjugated to an antibody, in a manner that will allowtheir targeting, internalization, release or presentation to bloodcomponents at the site of the targeted tumor cells as required usingknown conjugation technology (See, e.g., Ghose et al., Methods Enzymol.,93:280 [1983]).

For example, in some embodiments the present invention providesimmunotoxins targeted at stem cell cancer marker of the presentinvention. Immunotoxins are conjugates of a specific targeting agenttypically a tumor-directed antibody or fragment, with a cytotoxic agent,such as a toxin moiety. The targeting agent directs the toxin to, andthereby selectively kills, cells carrying the targeted antigen. In someembodiments, therapeutic antibodies employ crosslinkers that providehigh in vivo stability (Thorpe et al., Cancer Res., 48:6396 [1988]).

In other embodiments, particularly those involving treatment of solidtumors, antibodies are designed to have a cytotoxic or otherwiseanticellular effect against the tumor vasculature, by suppressing thegrowth or cell division of the vascular endothelial cells. This attackis intended to lead to a tumor-localized vascular collapse, deprivingthe tumor cells, particularly those tumor cells distal of thevasculature, of oxygen and nutrients, ultimately leading to cell deathand tumor necrosis.

In some embodiments, antibody based therapeutics are formulated aspharmaceutical compositions as described below. In some embodiments,administration of an antibody composition of the present inventionresults in a measurable decrease in cancer (e.g., decrease orelimination of tumor).

VI. Therapeutic Compositions and Administration

A pharmaceutical composition containing a regulator of tumorigenesisaccording the present invention can be administered by any effectivemethod. For example, an IL8-CXCR1 signaling pathway antagonist, or othertherapeutic agent that acts as an antagonist of proteins in theIL8-CXCR1 signal transduction/response pathway can be administered byany effective method. In certain embodiments of the present invention,the therapeutic agent comprises Repertaxin or a derivative thereof.

In certain embodiments, a physiologically appropriate solutioncontaining an effective concentration of an IL8-CXCR1 signaling pathwayantagonist can be administered topically, intraocularly, parenterally,orally, intranasally, intravenously, intramuscularly, subcutaneously orby any other effective means. In particular, the IL8-CXCR1 signalingpathway antagonist agent may be directly injected into a target canceror tumor (e.g., into breast tissue) by a needle in amounts effective totreat the tumor cells of the target tissue. Alternatively, a cancer ortumor present in a body cavity such as in the eye, gastrointestinaltract, genitourinary tract (e.g., the urinary bladder), pulmonary andbronchial system and the like can receive a physiologically appropriatecomposition (e.g., a solution such as a saline or phosphate buffer, asuspension, or an emulsion, which is sterile) containing an effectiveconcentration of an IL8-CXCR1 signaling pathway antagonist via directinjection with a needle or via a catheter or other delivery tube placedinto the cancer or tumor afflicted hollow organ. Any effective imagingdevice such as X-ray, sonogram, or fiber-optic visualization system maybe used to locate the target tissue and guide the needle or cathetertube. In another alternative, a physiologically appropriate solutioncontaining an effective concentration of an IL8-CXCR1 signaling pathwayantagonist can be administered systemically into the blood circulationto treat a cancer or tumor that cannot be directly reached oranatomically isolated.

Such manipulations have in common the goal of placing the IL8-CXCR1signaling pathway antagonist in sufficient contact with the target tumorto permit the antagonist to contact, transduce or transfect the tumorcells (depending on the nature of the agent). In one embodiment, solidtumors present in the epithelial linings of hollow organs may be treatedby infusing the suspension into a hollow fluid filled organ, or byspraying or misting into a hollow air filled organ. Thus, the tumorcells (such as a solid tumor stem cells) may be present in or among theepithelial tissue in the lining of pulmonary bronchial tree, the liningof the gastrointestinal tract, the lining of the female reproductivetract, genitourinary tract, bladder, the gall bladder and any otherorgan tissue accessible to contact with the IL8-CXCR1 signaling pathwayantagonist. In another embodiment, the solid tumor may be located in oron the lining of the central nervous system, such as, for example, thespinal cord, spinal roots or brain, so that the IL8-CXCR1 signalingpathway antagonist infused in the cerebrospinal fluid contacts andtransduces the cells of the solid tumor in that space. One skilled inthe art of oncology can appreciate that the antagonist can beadministered to the solid tumor by direct injection into the tumor sothat the antagonist contacts and affects the tumor cells inside thetumor.

The tumorigenic cells identified by the present invention can also beused to raise anti-cancer cell antibodies. In one embodiment, the methodinvolves obtaining an enriched population of tumorigenic cells orisolated tumorigenic cells; treating the population to prevent cellreplication (for example, by irradiation); and administering the treatedcell to a human or animal subject in an amount effective for inducing animmune response to solid tumor stem cells. For guidance as to aneffective dose of cells to be injected or orally administered; see, U.S.Pat. Nos. 6,218,166, 6,207,147, and 6,156,305, incorporated herein byreference. In another embodiment, the method involves obtaining anenriched population of solid tumor stem cells or isolated solid tumorstem cells; mixing the tumor stem cells in an in vitro culture withimmune effector cells (according to immunological methods known in theart) from a human subject or host animal in which the antibody is to beraised; removing the immune effector cells from the culture; andtransplanting the immune effector cells into a host animal in a dosethat is effective to stimulate an immune response in the animal.

In some embodiments of the present invention, the anti-tumorigenictherapeutic agents (e.g. IL8-CXCR1 signaling pathway antagonists) of thepresent invention are co-administered with other anti-neoplastictherapies. A wide range of therapeutic agents find use with the presentinvention. Any therapeutic agent that can be co-administered with theagents of the present invention, or associated with the agents of thepresent invention is suitable for use in the methods of the presentinvention.

Various classes of antineoplastic (e.g., anticancer) agents arecontemplated for use in certain embodiments of the present invention.Anticancer agents suitable for use with the present invention include,but are not limited to, agents that induce apoptosis, agents thatinhibit adenosine deaminase function, inhibit pyrimidine biosynthesis,inhibit purine ring biosynthesis, inhibit nucleotide interconversions,inhibit ribonucleotide reductase, inhibit thymidine monophosphate (TMP)synthesis, inhibit dihydrofolate reduction, inhibit DNA synthesis, formadducts with DNA, damage DNA, inhibit DNA repair, intercalate with DNA,deaminate asparagines, inhibit RNA synthesis, inhibit protein synthesisor stability, inhibit microtubule synthesis or function, and the like.

In some embodiments, exemplary anticancer agents suitable for use incompositions and methods of the present invention include, but are notlimited to: 1) alkaloids, including microtubule inhibitors (e.g.,vincristine, vinblastine, and vindesine, etc.), microtubule stabilizers(e.g., paclitaxel (TAXOL), and docetaxel, etc.), and chromatin functioninhibitors, including topoisomerase inhibitors, such asepipodophyllotoxins (e.g., etoposide (VP-16), and teniposide (VM-26),etc.), and agents that target topoisomerase I (e.g., camptothecin andisirinotecan (CPT-11), etc.); 2) covalent DNA-binding agents (alkylatingagents), including nitrogen mustards (e.g., mechlorethamine,chlorambucil, cyclophosphamide, ifosphamide, and busulfan (MYLERAN),etc.), nitrosoureas (e.g., carmustine, lomustine, and semustine, etc.),and other alkylating agents (e.g., dacarbazine, hydroxymethylmelamine,thiotepa, and mitomycin, etc.); 3) noncovalent DNA-binding agents(antitumor antibiotics), including nucleic acid inhibitors (e.g.,dactinomycin (actinomycin D), etc.), anthracyclines (e.g., daunorubicin(daunomycin, and cerubidine), doxorubicin (adriamycin), and idarubicin(idamycin), etc.), anthracenediones (e.g., anthracycline analogues, suchas mitoxantrone, etc.), bleomycins (BLENOXANE), etc., and plicamycin(mithramycin), etc.; 4) antimetabolites, including antifolates (e.g.,methotrexate, FOLEX, and MEXATE, etc.), purine antimetabolites (e.g.,6-mercaptopurine (6-MP, PURINETHOL), 6-thioguanine (6-TG), azathioprine,acyclovir, ganciclovir, chlorodeoxyadenosine, 2-chlorodeoxyadenosine(CdA), and 2′-deoxycoformycin (pentostatin), etc.), pyrimidineantagonists (e.g., fluoropyrimidines (e.g., 5-fluorouracil (ADRUCIL),5-fluorodeoxyuridine (FdUrd) (floxuridine)) etc.), and cytosinearabinosides (e.g., CYTOSAR (ara-C) and fludarabine, etc.); 5) enzymes,including L-asparaginase, and hydroxyurea, etc.; 6) hormones, includingglucocorticoids, antiestrogens (e.g., tamoxifen, etc.), nonsteroidalantiandrogens (e.g., flutamide, etc.), and aromatase inhibitors (e.g.,anastrozole (ARIMIDEX), etc.); 7) platinum compounds (e.g., cisplatinand carboplatin, etc.); 8) monoclonal antibodies conjugated withanticancer drugs, toxins, and/or radionuclides, etc.; 9) biologicalresponse modifiers (e.g., interferons (e.g., IFN-α, etc.) andinterleukins (e.g., IL-2, etc.), etc.); 10) adoptive immunotherapy; 11)hematopoietic growth factors; 12) agents that induce tumor celldifferentiation (e.g., all-trans-retinoic acid, etc.); 13) gene therapytechniques; 14) antisense therapy techniques; 15) tumor vaccines; 16)therapies directed against tumor metastases (e.g., batimastat, etc.);17) angiogenesis inhibitors; 18) proteosome inhibitors (e.g., VELCADE);19) inhibitors of acetylation and/or methylation (e.g., HDACinhibitors); 20) modulators of NF kappa B; 21) inhibitors of cell cycleregulation (e.g., CDK inhibitors); 22) modulators of p53 proteinfunction; and 23) radiation.

Any oncolytic agent that is routinely used in a cancer therapy contextfinds use in the compositions and methods of the present invention. Forexample, the U.S. Food and Drug Administration maintains a formulary ofoncolytic agents approved for use in the United States. Internationalcounterpart agencies to the U.S.F.D.A. maintain similar formularies.Table 3 provides a list of exemplary antineoplastic agents approved foruse in the U.S. Those skilled in the art will appreciate that the“product labels” required on all U.S. approved chemotherapeuticsdescribe approved indications, dosing information, toxicity data, andthe like, for the exemplary agents.

TABLE 3 Aldesleukin Proleukin Chiron Corp., (des-alanyl-1, serine-125human interleukin-2) Emeryville, CA Alemtuzumab Campath Millennium and(IgG1κ anti CD52 antibody) ILEX Partners, LP, Cambridge, MA AlitretinoinPanretin Ligand (9-cis-retinoic acid) Pharmaceuticals, Inc., San DiegoCA Allopurinol Zyloprim GlaxoSmithKline,(1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one Research Trianglemonosodium salt) Park, NC Altretamine Hexalen US Bioscience, West(N,N,N′,N′,N″,N″,-hexamethyl-1,3,5-triazine-2,4, Conshohocken, PA6-triamine) Amifostine Ethyol US Bioscience (ethanethiol,2-[(3-aminopropyl)amino]-, dihydrogen phosphate (ester)) AnastrozoleArimidex AstraZeneca (1,3-Benzenediacetonitrile, a,a,a′,a′-tetramethyl-Pharmaceuticals, LP, 5-(1H-1,2,4-triazol-1-ylmethyl)) Wilmington, DEArsenic trioxide Trisenox Cell Therapeutic, Inc., Seattle, WAAsparaginase Elspar Merck & Co., Inc., (L-asparagine amidohydrolase,type EC-2) Whitehouse Station, NJ BCG Live TICE BCG Organon Teknika,(lyophilized preparation of an attenuated strain of Corp., Durham, NCMycobacterium bovis (Bacillus Calmette-Gukin [BCG], substrain Montreal)bexarotene capsules Targretin Ligand(4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2- Pharmaceuticalsnapthalenyl) ethenyl] benzoic acid) bexarotene gel Targretin LigandPharmaceuticals Bleomycin Blenoxane Bristol-Myers Squibb (cytotoxicglycopeptide antibiotics produced by Co., NY, NY Streptomycesverticillus; bleomycin A₂ and bleomycin B₂) Capecitabine Xeloda Roche(5′-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]- cytidine) CarboplatinParaplatin Bristol-Myers Squibb (platinum, diammine [1,1-cyclobutanedicarboxylato(2-)-0,0′]-,(SP-4-2)) Carmustine BCNU, BiCNUBristol-Myers Squibb (1,3-bis(2-chloroethyl)-1-nitrosourea) Carmustinewith Polifeprosan 20 Implant Gliadel Wafer Guilford Pharmaceuticals,Inc., Baltimore, MD Celecoxib Celebrex Searle (as4-[5-(4-methylphenyl)-3-(trifluoromethyl)- Pharmaceuticals,1H-pyrazol-1-yl] England benzenesulfonamide) Chlorambucil LeukeranGlaxoSmithKline (4-[bis(2chlorethyl)amino]benzenebutanoic acid)Cisplatin Platinol Bristol-Myers Squibb (PtCl₂H₆N₂) CladribineLeustatin, 2-CdA R.W. Johnson (2-chloro-2′-deoxy-b-D-adenosine)Pharmaceutical Research Institute, Raritan, NJ Cyclophosphamide Cytoxan,Neosar Bristol-Myers Squibb (2-[bis(2-chloroethyl)amino]tetrahydro-2H-13,2- oxazaphosphorine 2-oxide monohydrate) CytarabineCytosar-U Pharmacia & Upjohn (1-b-D-Arabinofuranosylcytosine, C₉H₁₃N₃O₅)Company cytarabine liposomal DepoCyt Skye Pharmaceuticals, Inc., SanDiego, CA Dacarbazine DTIC-Dome Bayer AG,(5-(3,3-dimethyl-1-triazeno)-imidazole-4- Leverkusen, carboxamide(DTIC)) Germany Dactinomycin, actinomycin D Cosmegen Merck (actinomycinproduced by Streptomyces parvullus, C₆₂H₈₆N₁₂O₁₆) Darbepoetin alfaAranesp Amgen, Inc., (recombinant peptide) Thousand Oaks, CAdaunorubicin liposomal DanuoXome Nexstar((8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a- Pharmaceuticals, Inc.,L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro- Boulder, CO6,8,11-trihydroxy-1-methoxy-5,12- naphthacenedione hydrochloride)Daunorubicin HCl, daunomycin Cerubidine Wyeth Ayerst,((1S,3S)-3-Acetyl-1,2,3,4,6,11-hexahydro- Madison, NJ3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1- naphthacenyl3-amino-2,3,6-trideoxy-(alpha)-L- lyxo-hexopyranoside hydrochloride)Denileukin diftitox Ontak Seragen, Inc., (recombinant peptide)Hopkinton, MA Dexrazoxane Zinecard Pharmacia & Upjohn((S)-4,4′-(1-methyl-1,2-ethanediyl)bis-2,6- Company piperazinedione)Docetaxel Taxotere Aventis ((2R,3S)-N-carboxy-3-phenylisoserine, N-tert-Pharmaceuticals, Inc., butyl ester, 13-ester with 5b-20-epoxy-Bridgewater, NJ 12a,4,7b,10b,13a-hexahydroxytax-11-en-9-one 4- acetate2-benzoate, trihydrate) Doxorubicin HCl Adriamycin, Pharmacia & Upjohn(8S,10S)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo- Rubex Companyhexopyranosyl)oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12- naphthacenedionehydrochloride) doxorubicin Adriamycin PFS Pharmacia & Upjohn IntravenousCompany injection doxorubicin liposomal Doxil Sequus Pharmaceuticals,Inc., Menlo park, CA dromostanolone propionate Dromostanolone Eli Lilly& Company, (17b-Hydroxy-2a-methyl-5a-androstan-3-one Indianapolis, INpropionate) dromostanolone propionate Masterone Syntex, Corp., Paloinjection Alto, CA Elliott's B Solution Elliott's B Orphan Medical, IncSolution Epirubicin Ellence Pharmacia & Upjohn((8S-cis)-10-[(3-amino-2,3,6-trideoxy-a-L- Companyarabino-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy- 5,12-naphthacenedionehydrochloride) Epoetin alfa Epogen Amgen, Inc (recombinant peptide)Estramustine Emcyt Pharmacia & Upjohn(estra-1,3,5(10)-triene-3,17-diol(17(beta))-, 3- Company[bis(2-chloroethyl)carbamate] 17-(dihydrogen phosphate), disodium salt,monohydrate, or estradiol 3-[bis(2-chloroethyl)carbamate] 17-(dihydrogen phosphate), disodium salt, monohydrate) Etoposide phosphateEtopophos Bristol-Myers Squibb (4′-Demethylepipodophyllotoxin9-[4,6-O—(R)- ethylidene-(beta)-D-glucopyranoside], 4′- (dihydrogenphosphate)) etoposide, VP-16 Vepesid Bristol-Myers Squibb(4′-demethylepipodophyllotoxin 9-[4,6-0-(R)-ethylidene-(beta)-D-glucopyranoside]) Exemestane Aromasin Pharmacia &Upjohn (6-methylenandrosta-1,4-diene-3,17-dione) Company FilgrastimNeupogen Amgen, Inc (r-metHuG-CSF) floxuridine (intraarterial) FUDRRoche (2′-deoxy-5-fluorouridine) Fludarabine Fludara BerlexLaboratories, (fluorinated nucleotide analog of the antiviral Inc.,Cedar Knolls, agent vidarabine, 9-b-D-arabinofuranosyladenine NJ(ara-A)) Fluorouracil, 5-FU Adrucil ICN Pharmaceuticals,(5-fluoro-2,4(1H,3H)-pyrimidinedione) Inc., Humacao, Puerto RicoFulvestrant Faslodex IPR Pharmaceuticals, (7-alpha-[9-(4,4,5,5,5-pentafluoropentylsulphinyl) Guayama, Puertononyl]estra-1,3,5-(10)-triene-3,17-beta-diol) Rico Gemcitabine GemzarEli Lilly (2′-deoxy-2′,2′-difluorocytidine monohydrochloride (b-isomer))Gemtuzumab Ozogamicin Mylotarg Wyeth Ayerst (anti-CD33 hP67.6) Goserelinacetate Zoladex Implant AstraZeneca (acetate salt of[D-Ser(But)⁶,Azgly¹⁰]LHRH; pyro- PharmaceuticalsGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro- Azgly-NH2 acetate[C₅₉H₈₄N₁₈O₁₄•(C₂H₄O₂)_(x) Hydroxyurea Hydrea Bristol-Myers SquibbIbritumomab Tiuxetan Zevalin Biogen IDEC, Inc., (immunoconjugateresulting from a thiourea Cambridge MA covalent bond between themonoclonal antibody Ibritumomab and the linker-chelator tiuxetan [N-[2-bis(carboxymethyl)amino]-3-(p- isothiocyanatophenyl)-propyl]-[N-[2-bis(carboxymethyl)amino]-2-(methyl)- ethyl]glycine) Idarubicin IdamycinPharmacia & Upjohn (5,12-Naphthacenedione, 9-acetyl-7-[(3-amino- Company2,3,6-trideoxy-(alpha)-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,9,11- trihydroxyhydrochloride,(7S-cis)) Ifosfamide IFEX Bristol-Myers Squibb (3-(2-chloroethyl)-2-[(2-chloroethyl)amino]tetrahydro-2H-1,3,2- oxazaphosphorine 2-oxide)Imatinib Mesilate Gleevec Novartis AG, Basel,(4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl- Switzerland3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]- phenyl]benzamidemethanesulfonate) Interferon alfa-2a Roferon-A Hoffmann-La Roche,(recombinant peptide) Inc., Nutley, NJ Interferon alfa-2b Intron ASchering AG, Berlin, (recombinant peptide) (Lyophilized GermanyBetaseron) Irinotecan HCl Camptosar Pharmacia & Upjohn((4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbonyloxy]-Company 1H-pyrano[3′,4′:6, 7] indolizino[1,2-b] quinoline-3,14(4H,12H)dione hydrochloride trihydrate) Letrozole Femara Novartis(4,4′-(1H-1,2,4-Triazol-1-ylmethylene) dibenzonitrile) LeucovorinWellcovorin, Immunex, Corp., (L-Glutamic acid, N[4[[(2amino-5-formyl-Leucovorin Seattle, WA 1,4,5,6,7,8 hexahydro4oxo6-pteridinyl)methyl]amino]benzoyl], calcium salt (1:1)) Levamisole HClErgamisol Janssen Research ((−)-(S)-2,3,5,6-tetrahydro-6-phenylimidazo[2,1- Foundation, b] thiazole monohydrochloride C₁₁H₁₂N₂S•HCl)Titusville, NJ Lomustine CeeNU Bristol-Myers Squibb(1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea) Meclorethamine, nitrogenmustard Mustargen Merck (2-chloro-N-(2-chloroethyl)-N-methylethanaminehydrochloride) Megestrol acetate Megace Bristol-Myers Squibb17α(acetyloxy)-6-methylpregna-4,6-diene- 3,20-dione Melphalan, L-PAMAlkeran GlaxoSmithKline (4-[bis(2-chloroethyl) amino]-L-phenylalanine)Mercaptopurine, 6-MP Purinethol GlaxoSmithKline(1,7-dihydro-6H-purine-6-thione monohydrate) Mesna Mesnex Asta Medica(sodium 2-mercaptoethane sulfonate) Methotrexate Methotrexate LederleLaboratories (N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L- glutamic acid) MethoxsalenUvadex Therakos, Inc., Way(9-methoxy-7H-furo[3,2-g][1]-benzopyran-7-one) Exton, Pa Mitomycin CMutamycin Bristol-Myers Squibb mitomycin C Mitozytrex SuperGen, Inc.,Dublin, CA Mitotane Lysodren Bristol-Myers Squibb(1,1-dichloro-2-(o-chlorophenyl)-2-(p- chlorophenyl) ethane)Mitoxantrone Novantrone Immunex (1,4-dihydroxy-5,8-bis[[2-[(2-Corporation hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedionedihydrochloride) Nandrolone phenpropionate Durabolin-50 Organon, Inc.,West Orange, NJ Nofetumomab Verluma Boehringer Ingelheim Pharma KG,Germany Oprelvekin Neumega Genetics Institute, (IL-11) Inc., Alexandria,VA Oxaliplatin Eloxatin Sanofi Synthelabo,(cis-[(1R,2R)-1,2-cyclohexanediamine-N,N′] Inc., NY, NY[oxalato(2-)-O,O′] platinum) Paclitaxel TAXOL Bristol-Myers Squibb(5β,20-Epoxy-1,2a,4,7β,10β,13a- hexahydroxytax-11-en-9-one4,10-diacetate 2- benzoate 13-ester with (2R,3S)-N-benzoyl-3-phenylisoserine) Pamidronate Aredia Novartis (phosphonic acid(3-amino-1-hydroxypropylidene) bis-, disodium salt, pentahydrate, (APD))Pegademase Adagen Enzon ((monomethoxypolyethylene glycol succinimidyl)(Pegademase Pharmaceuticals, Inc., 11-17-adenosine deaminase) Bovine)Bridgewater, NJ Pegaspargase Oncaspar Enzon (monomethoxypolyethyleneglycol succinimidyl L-asparaginase) Pegfilgrastim Neulasta Amgen, Inc(covalent conjugate of recombinant methionyl human G-CSF (Filgrastim)and monomethoxypolyethylene glycol) Pentostatin Nipent Parke-DavisPharmaceutical Co., Rockville, MD Pipobroman Vercyte AbbottLaboratories, Abbott Park, IL Plicamycin, Mithramycin Mithracin Pfizer,Inc., NY, NY (antibiotic produced by Streptomyces plicatus) Porfimersodium Photofrin QLT Phototherapeutics, Inc., Vancouver, CanadaProcarbazine Matulane Sigma Tau(N-isopropyl-μ-(2-methylhydrazino)-p-toluamide Pharmaceuticals, Inc.,monohydrochloride) Gaithersburg, MD Quinacrine Atabrine Abbott Labs(6-chloro-9-(1-methyl-4-diethyl-amine) butylamino-2-methoxyacridine)Rasburicase Elitek Sanofi-Synthelabo, (recombinant peptide) Inc.,Rituximab Rituxan Genentech, Inc., (recombinant anti-CD20 antibody)South San Francisco, CA Sargramostim Prokine Immunex Corp (recombinantpeptide) Streptozocin Zanosar Pharmacia & Upjohn (streptozocin2-deoxy-2- Company [[(methylnitrosoamino)carbonyl]amino]-a(and b)-D-glucopyranose and 220 mg citric acid anhydrous) Talc Sclerosol Bryan,Corp., (Mg₃Si₄O₁₀(OH)₂) Woburn, MA Tamoxifen Nolvadex AstraZeneca((Z)2-[4-(1,2-diphenyl-1-butenyl) phenoxy]-N,N- Pharmaceuticalsdimethylethanamine 2-hydroxy-1,2,3- propanetricarboxylate (1:1))Temozolomide Temodar Schering(3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as- tetrazine-8-carboxamide)teniposide, VM-26 Vumon Bristol-Myers Squibb(4′-demethylepipodophyllotoxin 9-[4,6-0-(R)-2-thenylidene-(beta)-D-glucopyranoside]) Testolactone Teslac Bristol-MyersSquibb (13-hydroxy-3-oxo-13,17-secoandrosta-1,4-dien- 17-oic acid[dgr]-lactone) Thioguanine, 6-TG Thioguanine GlaxoSmithKline(2-amino-1,7-dihydro-6H-purine-6-thione) Thiotepa Thioplex Immunex(Aziridine, 1,1′,1″-phosphinothioylidynetris-, or Corporation Tris(1-aziridinyl) phosphine sulfide) Topotecan HCl Hycamtin GlaxoSmithKline((S)-10-[(dimethylamino) methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7] indolizino [1,2-b]quinoline-3,14-(4H,12H)-dione monohydrochloride) Toremifene FarestonRoberts (2-(p-[(Z)-4-chloro-1,2-diphenyl-1-butenyl]- Pharmaceuticalphenoxy)-N,N-dimethylethylamine citrate (1:1)) Corp., Eatontown, NJTositumomab, I 131 Tositumomab Bexxar Corixa Corp., Seattle,(recombinant murine immunotherapeutic WA monoclonal IgG_(2a) lambdaanti-CD20 antibody (I 131 is a radioimmunotherapeutic antibody))Trastuzumab Herceptin Genentech, Inc (recombinant monoclonal IgG₁ kappaanti-HER2 antibody) Tretinoin, ATRA Vesanoid Roche (all-trans retinoicacid) Uracil Mustard Uracil Mustard Roberts Labs Capsules Valrubicin,N-trifluoroacetyladriamycin-14- Valstar Anthra --> Medeva valerate((2S-cis)-2-[1,2,3,4,6,11-hexahydro-2,5,12- trihydroxy-7methoxy-6,11-dioxo-[[4 2,3,6-trideoxy-3-[(trifluoroacetyl)-amino-α-L-lyxo-hexopyranosyl]oxyl]-2-naphthacenyl]-2-oxoethyl pentanoate) Vinblastine,Leurocristine Velban Eli Lilly (C₄₆H₅₆N₄O₁₀•H₂SO₄) Vincristine OncovinEli Lilly (C₄₆H₅₆N₄O₁₀•H₂SO₄) Vinorelbine Navelbine GlaxoSmithKline(3′,4′-didehydro-4′-deoxy-C′- norvincaleukoblastine [R-(R*,R*)-2,3-dihydroxybutanedioate (1:2)(salt)]) Zoledronate, Zoledronic acid ZometaNovartis ((1-Hydroxy-2-imidazol-1-yl-phosphonoethyl) phosphonic acidmonohydrate)

Antimicrobial therapeutic agents may also be used as therapeutic agentsin the present invention. Any agent that can kill, inhibit, or otherwiseattenuate the function of microbial organisms may be used, as well asany agent contemplated to have such activities. Antimicrobial agentsinclude, but are not limited to, natural and synthetic antibiotics,antibodies, inhibitory proteins (e.g., defensins), antisense nucleicacids, membrane disruptive agents and the like, used alone or incombination. Indeed, any type of antibiotic may be used including, butnot limited to, antibacterial agents, antiviral agents, antifungalagents, and the like.

In still further embodiments, the present invention provides compoundsof the present invention (and any other chemotherapeutic agents)associated with targeting agents that are able to specifically targetparticular cell types (e.g., tumor cells). Generally, the therapeuticcompound that is associated with a targeting agent, targets neoplasticcells through interaction of the targeting agent with a cell surfacemoiety that is taken into the cell through receptor mediatedendocytosis.

Any moiety known to be located on the surface of target cells (e.g.,tumor cells) finds use with the present invention. For example, anantibody directed against such a moiety targets the compositions of thepresent invention to cell surfaces containing the moiety. Alternatively,the targeting moiety may be a ligand directed to a receptor present onthe cell surface or vice versa. Similarly, vitamins also may be used totarget the therapeutics of the present invention to a particular cell.

As used herein, the term “targeting molecules” refers to chemicalmoieties, and portions thereof useful for targeting therapeuticcompounds to cells, tissues, and organs of interest. Various types oftargeting molecules are contemplated for use with the present inventionincluding, but not limited to, signal peptides, antibodies, nucleicacids, toxins and the like. Targeting moieties may additionally promotethe binding of the associated chemical compounds (e.g., small molecules)or the entry of the compounds into the targeted cells, tissues, andorgans. Preferably, targeting moieties are selected according to theirspecificity, affinity, and efficacy in selectively delivering attachedcompounds to targeted sites within a subject, tissue, or a cell,including specific subcellular locations and organelles.

Various efficiency issues affect the administration of all drugs—and ofhighly cytotoxic drugs (e.g., anticancer drugs) in particular. One issueof particular importance is ensuring that the administered agents affectonly targeted cells (e.g., cancer cells), tissues, or organs. Thenonspecific or unintended delivery of highly cytotoxic agents tonontargeted cells can cause serious toxicity issues.

Numerous attempts have been made to devise drug-targeting schemes toaddress the problems associated with nonspecific drug delivery. (Seee.g., K. N. Syrigos and A. A. Epenetos Anticancer Res., 19:606-614(1999); Y. J. Park et al., J. Controlled Release, 78:67-79 (2002); R. V.J. Chari, Adv. Drug Deliv. Rev., 31:89-104 (1998); and D. Putnam and J.Kopecek, Adv. Polymer Sci., 122:55-123 (1995)). Conjugating targetingmoieties such as antibodies and ligand peptides (e.g., RDG forendothelium cells) to drug molecules has been used to alleviate somecollateral toxicity issues associated with particular drugs.

The compounds and anticancer agents may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. In some embodiments, thepharmaceutical compositions of the present invention may contain oneagent (e.g., an antibody). In other embodiments, the pharmaceuticalcompositions contain a mixture of at least two agents (e.g., an antibodyand one or more conventional anticancer agents). In still furtherembodiments, the pharmaceutical compositions of the present inventioncontain at least two agents that are administered to a patient under oneor more of the following conditions: at different periodicities, atdifferent durations, at different concentrations, by differentadministration routes, etc. In some embodiments, the IL8-CXCR1 signalingpathway antagonist is administered prior to the second anticancer agent,e.g., 0.5, 1, 2 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days,1, 2, 3, or 4 weeks prior to the administration of the anticancer agent.In some embodiments, the IL8-CXCR1 signaling pathway antagonist isadministered after the second anticancer agent, e.g., 0.5, 1, 2 3, 4, 5,10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, 1, 2, 3, or 4 weeks afterthe administration of the anticancer agent. In some embodiments, theIL8-CXCR1 signaling pathway antagonist and the second anticancer agentare administered concurrently but on different schedules, e.g., theIL8-CXCR1 signaling pathway antagonist compound is administered dailywhile the second anticancer agent is administered once a week, onceevery two weeks, once every three weeks, or once every four weeks. Inother embodiments, the IL8-CXCR1 signaling pathway antagonist isadministered once a week while the second anticancer agent isadministered daily, once a week, once every two weeks, once every threeweeks, or once every four weeks.

Depending on the condition being treated, preferred embodiments of thepresent pharmaceutical compositions are formulated and administeredsystemically or locally. Techniques for formulation and administrationcan be found in the latest edition of “Remington's PharmaceuticalSciences” (Mack Publishing Co, Easton Pa.). Suitable routes may, forexample, include oral or transmucosal administration as well asparenteral delivery (e.g., intramuscular, subcutaneous, intramedullary,intrathecal, intraventricular, intravenous, intraperitoneal, orintranasal administration).

The present invention contemplates administering therapeutic compoundsand, in some embodiments, one or more conventional anticancer agents, inaccordance with acceptable pharmaceutical delivery methods andpreparation techniques. For example, therapeutic compounds and suitableanticancer agents can be administered to a subject intravenously in apharmaceutically acceptable carrier such as physiological saline.Standard methods for intracellular delivery of pharmaceutical agents arecontemplated (e.g., delivery via liposome). Such methods are well knownto those of ordinary skill in the art.

In some embodiments, the formulations of the present invention areuseful for parenteral administration (e.g., intravenous, subcutaneous,intramuscular, intramedullary, and intraperitoneal). Therapeuticco-administration of some contemplated anticancer agents (e.g.,therapeutic polypeptides) can also be accomplished using gene therapyreagents and techniques.

In some embodiments of the present invention, therapeutic compounds areadministered to a subject alone, or in combination with one or moreconventional anticancer agents (e.g., nucleotide sequences, drugs,hormones, etc.) or in pharmaceutical compositions where the componentsare optionally mixed with excipient(s) or other pharmaceuticallyacceptable carriers. In preferred embodiments of the present invention,pharmaceutically acceptable carriers are biologically inert. Inpreferred embodiments, the pharmaceutical compositions of the presentinvention are formulated using pharmaceutically acceptable carriers wellknown in the art in dosages suitable for oral administration. Suchcarriers enable the pharmaceutical compositions to be formulated astablets, pills, capsules, dragees, liquids, gels, syrups, slurries,solutions, suspensions and the like, for respective oral or nasalingestion by a subject.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipients, optionally grinding theresulting mixture, and processing the mixture into granules, afteradding suitable auxiliaries, if desired, to obtain tablets or drageecores. Suitable excipients are carbohydrate or protein fillers such assugars, including lactose, sucrose, mannitol, or sorbitol; starch fromcorn, wheat, rice, potato, etc.; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gumsincluding arabic and tragacanth; and proteins such as gelatin andcollagen. If desired, disintegrating or solubilizing agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, alginicacid or a salt thereof such as sodium alginate.

In preferred embodiments, dosing and administration regimes are tailoredby the clinician, or others skilled in the pharmacological arts, basedupon well known pharmacological and therapeutic considerationsincluding, but not limited to, the desired level of therapeutic effect,and the practical level of therapeutic effect obtainable. Generally, itis advisable to follow well-known pharmacological principles foradministrating chemotherapeutic agents (e.g., it is generally advisableto not change dosages by more than 50% at time and no more than every3-4 agent half-lives). For compositions that have relatively little orno dose-related toxicity considerations, and where maximum efficacy(e.g., destruction of cancer cells) is desired, doses in excess of theaverage required dose are not uncommon. This approach to dosing iscommonly referred to as the “maximal dose” strategy. In certainembodiments, the IL8-CXCR1 signaling pathway antagonist is administeredto a subject at a dose of 1-40 mg per day (e.g. for 4-6 weeks). Incertain embodiments, subject is administered a loading dose of between15-70 mg of the IL8-CXCR1 signaling pathway antagonist. In certainembodiments, the subject is administered a loading dose of about 35-45mg of the IL8-CXCR1 signaling pathway antagonist (e.g. subcutaneously),and then daily doses of about 10 mg (e.g. subcutaneously) for about 4-6weeks.

Additional dosing considerations relate to calculating proper targetlevels for the agent being administered, the agent's accumulation andpotential toxicity, stimulation of resistance, lack of efficacy, anddescribing the range of the agent's therapeutic index.

In certain embodiments, the present invention contemplates using routinemethods of titrating the agent's administration. One common strategy forthe administration is to set a reasonable target level for the agent inthe subject. In some preferred embodiments, agent levels are measured inthe subject's plasma. Proper dose levels and frequencies are thendesigned to achieve the desired steady-state target level for the agent.Actual, or average, levels of the agent in the subject are monitored(e.g., hourly, daily, weekly, etc.) such that the dosing levels orfrequencies can be adjusted to maintain target levels. Of course, thepharmacokinetics and pharmacodynamics (e.g., bioavailability, clearanceor bioaccumulation, biodistribution, drug interactions, etc.) of theparticular agent or agents being administered can potentially impactwhat are considered reasonable target levels and thus impact dosinglevels or frequencies.

Target-level dosing methods typically rely upon establishing areasonable therapeutic objective defined in terms of a desirable range(or therapeutic range) for the agent in the subject. In general, thelower limit of the therapeutic range is roughly equal to theconcentration of the agent that provides about 50% of the maximumpossible therapeutic effect. The upper limit of the therapeutic range isusually established by the agent's toxicity and not by its efficacy. Thepresent invention contemplates that the upper limit of the therapeuticrange for a particular agent will be the concentration at which lessthan 5 or 10% of subjects exhibit toxic side effects. In someembodiments, the upper limit of the therapeutic range is about twotimes, or less, than the lower limit. Those skilled in the art willunderstand that these dosing consideration are highly variable and tosome extent individualistic (e.g., based on genetic predispositions,immunological considerations, tolerances, resistances, and the like).Thus, in some embodiments, effective target dosing levels for an agentin a particular subject may be 1, . . . 5, . . . 10, . . . 15, . . . 20,. . . 50, . . . 75, . . . 100, . . . 200, . . . X %, greater thanoptimal in another subject. Conversely, some subjects may suffersignificant side effects and toxicity related health issues at dosinglevels or frequencies far less (1, . . . 5, . . . 10, . . . 15, . . .20, . . . 50, . . . 75, . . . 100, . . . 200, . . . X %) than thosetypically producing optimal therapeutic levels in some or a majority ofsubjects. In the absence of more specific information, targetadministration levels are often set in the middle of the therapeuticrange.

In preferred embodiments, the clinician rationally designs anindividualized dosing regimen based on known pharmacological principlesand equations. In general, the clinician designs an individualizeddosing regimen based on knowledge of various pharmacological andpharmacokinetic properties of the agent, including, but not limited to,F (fractional bioavailability of the dose), Cp (concentration in theplasma), CL (clearance/clearance rate), Vss (volume of drug distributionat steady state) Css (concentration at steady state), and t½ (drughalf-life), as well as information about the agent's rate of absorptionand distribution. Those skilled in the art are referred to any number ofwell known pharmacological texts (e.g., Goodman and Gilman's,Pharmaceutical Basis of Therapeutics, 10th ed., Hardman et al., eds.,2001) for further explanation of these variables and for completeequations illustrating the calculation of individualized dosing regimes.Those skilled in the art also will be able to anticipate potentialfluctuations in these variables in individual subjects. For example, thestandard deviation in the values observed for F, CL, and Vss istypically about 20%, 50%, and 30%, respectively. The practical effect ofpotentially widely varying parameters in individual subjects is that 95%of the time the Css achieved in a subject is between 35 and 270% that ofthe target level. For drugs with low therapeutic indices, this is anundesirably wide range. Those skilled in the art will appreciate,however, that once the agent's Cp (concentration in the plasma) ismeasured, it is possible to estimate the values of F, CL, and Vssdirectly. This allows the clinician to effectively fine tune aparticular subject's dosing regimen.

In still other embodiments, the present invention contemplates thatcontinuing therapeutic drug monitoring techniques be used to furtheradjust an individual's dosing methods and regimens. For example, in oneembodiment, Css data is used is to further refine the estimates of CL/Fand to subsequently adjust the individual's maintenance dosing toachieve desired agent target levels using known pharmacologicalprinciples and equations. Therapeutic drug monitoring can be conductedat practically any time during the dosing schedule. In preferredembodiments, monitoring is carried out at multiple time points duringdosing and especially when administering intermittent doses. Forexample, drug monitoring can be conducted concomitantly, withinfractions of a second, seconds, minutes, hours, days, weeks, months,etc., of administration of the agent regardless of the dosingmethodology employed (e.g., intermittent dosing, loading doses,maintenance dosing, random dosing, or any other dosing method). However,those skilled in the art will appreciate that when sampling rapidlyfollows agent administration the changes in agent effects and dynamicsmay not be readily observable because changes in plasma concentration ofthe agent may be delayed (e.g., due to a slow rate of distribution orother pharmacodynamic factors). Accordingly, subject samples obtainedshortly after agent administration may have limited or decreased value.

The primary goal of collecting biological samples from the subjectduring the predicted steady-state target level of administration is tomodify the individual's dosing regimen based upon subsequentlycalculating revised estimates of the agent's CL/F ratio. However, thoseskilled in the art will appreciate that early postabsorptive drugconcentrations do not typically reflect agent clearance. Earlypostabsorptive drug concentrations are dictated principally by theagent's rate of absorption, the central, rather than the steady state,volume of agent distribution, and the rate of distribution. Each ofthese pharmacokinetic characteristics have limited value whencalculating therapeutic long-term maintenance dosing regimens.

Accordingly, in some embodiments, when the objective is therapeuticlong-term maintenance dosing, biological samples are obtained from thesubject, cells, or tissues of interest well after the previous dose hasbeen administered, and even more preferably shortly before the nextplanned dose is administered.

In still other embodiments, where the therapeutic agent is nearlycompletely cleared by the subject in the interval between doses, thenthe present invention contemplates collecting biological samples fromthe subject at various time points following the previousadministration, and most preferably shortly after the dose wasadministered.

VII. Repertaxin and Other Small Molecule CXCR1 Inhibitors

In certain embodiments, the methods, kits, and compositions of thepresent invention employ small molecule inhibitors of CXCR1. Oneexemplary agent is repartaxin. In certain embodiments, the in vivo doseof repartaxin is between 3 and 60 mg per kilogram (e.g., 3 . . . 30 . .. 50 . . . 60 mg/kg). In particular embodiments, the dose of repartaxinis about 30 mg per kilogram. The chemical formula for repartaxin isshown below:

In other embodiments, derivatives of the repertaxin are employed. Othersmall molecule CXCR1 antagonists include SB265610 (Glaxo SmithKlineBeecham; Benson et al., 2000, 151:196-197), as well as SCH 527123(2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methylfuran-2-yl)propyl]amino]-3,4-dioxocyclobut-1-enylamino}benzamide (SCH 527123), an orally bioavailable CXCR2/CXCR1 receptorantagonist (Schering Plough)). Other small molecule inhibitors can beidentified by the screening methods described above.

EXAMPLES

The following example is provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and is not to be construed as limiting the scope thereof.

Example 1 CXCR1 Identifies Cancer Stem Cells

This example describes the identification of CXCR1, as well as otherproteins (e.g., FBXO21), as cancer stem cell markers.

Cell Culture.

Breast cell lines (BCL) were obtained from the ATCC (“http://www.”followed by“lgcpromochem-atcc.com/common/catalog/cellBiology/cellBiologyIndex.cfm”)or from collections developed in the laboratories of Drs. S. Ethier (nowavailable on “http://www.” followed by“asterand.com/asterand/BIOREPOSITORY/hbreastcancercelllines.aspx, SUM44,SUM52, SUM149, SUM159, SUM185, SUM190, SUM225, SUM229”), V. J. Maus(BrCa-MZ-01), and V. Catros (S68). All BCLs tested were derived fromcarcinomas except MCF10A, which is derived from fibrocystic disease, andthe HMEC-derived 184A1, which was derived from normal mammary tissue.The cell lines were grown using the recommended culture conditions. Allexperiments were done with subconfluent cells in the exponential phaseof growth.

ALDEFLUOR Assay and Separation of the ALDH-Positive Population by FACS.

ALDH activity was assessed in 33 BCLs representing the main molecularsubtypes of human breast cancer. The ALDEFLUOR kit (StemCelltechnologies, Durham, N.C., USA) was used to isolate the population withhigh ALDH enzymatic activity (17). Cells obtained from subconfluent celllines after trypsinization or from freshly dissociated xenografts weresuspended in ALDEFLUOR assay buffer containing ALDH substrate (BAAA, 1μmol/l per 1×106 cells) and incubated for 40 minutes at 37° C. In eachexperiment a sample of cells was stained under identical conditions with50 mmol/L of diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor,as negative control. Flow cytometry sorting was conducted using aFACStarPLUS (Becton Dickinson). ALDEFLUOR Fluorescence was Excited at488 nm and Detected Using Standard Fluorescein Isothiocyanate (FITC)530/30 Band Pass Filter. For xenotransplanted tumors, incubation with ananti-H2Kd antibody (BD biosciences, 1/200, 20 min on ice) followed by asecondary antibody labeled with phycoerythrin (Jackson labs, 1/250, 20min on ice) was used to eliminate cells of mouse origin. The sortinggates were established using PI stained cells for viability,ALDEFLUOR-stained cells treated with DEAB, and those stained withsecondary antibody alone. Prior to RNA profiling or NOD/SCID miceinjection, the purity of sorted populations was checked using doublesorting of 10,000 ALDEFLUOR-positive and negative cells in BrCa-MZ-01and SUM159 cell lines. For both cell lines, sorted ALDEFLUOR-positivepopulations contained more than 98% of ALDEFLUOR-positive cells and noALDEFLUOR-positive cells were detected in the ALDEFLUOR-negativepopulation.

Tumorigenicity in NOD/SCID Mice.

Tumorigenicity of ALDEFLUOR-positive, -negative and unseparated SUM159,MDA-MB-453 and BrCa-MZ-01 cells was assessed in NOD/SCID mice. Fat padswere cleared of epithelium at 3 weeks of age prior to puberty andhumanized by injecting human fibroblasts (1:1 irradiated:non-irradiated,50,000 cells/1000 Matrigel/fat pad) as described (17). The animals wereeuthanized when the tumors were 1.2 cm in the largest diameter, incompliance with regulations for use of vertebrate animal in research. Aportion of each fat pad was fixed in formalin and embedded in paraffinfor histological analysis. Another portion was assessed by the ALDEFLUORassay, followed by sorting and serial transplantation.

Anchorage-Independent Culture.

ALDEFLUOR-positive, -negative and unseparated cells from 184A1, SUM149and SUM159 were plated as single cells in ultra-low attachment plates(Corning, Acton, Mass.) at low density (5000 viable cells/ml). Cellswere grown in serum-free mammary epithelial basal medium (Cambrex BioScience, Walkerville, Md.) for 3-7 days, as described (18). The capacityof cells to form spheres was quantified after treatment with differentdoses of IL8 (GenWay Biotech, San Diego, Calif.) added to the medium.

RNA Extraction.

Total RNA was extracted from frozen ALDEFLUOR-positive and -negativecells using DNA/RNA All Prep Maxi Kit, according to the manufacturer'sinstructions (Qiagen, Sample and Assay technologies, The Netherlands).Eight BCLs were used for transcriptional analysis: 184A1, BrCa-MZ-01,HCC1954, MDA-MB-231, MDA-MB-453, SK-BR-7, SUM149, and SUM159. RNAintegrity was controlled by denaturing formaldehyde agarose gelelectrophoresis and micro-analysis (Agilent Bioanalyzer, Palo Alto,Calif.).

Gene Expression Profiling with DNA Microarrays.

Gene expression analyses used Affymetrix U133 Plus 2.0 humanoligonucleotide microarrays containing over 47,000 transcripts andvariants including 38,500 well-characterized human genes. Preparation ofcRNA, hybridizations, washes and detection were done as recommended bythe supplier (“http://www.” followed by “affymetrix.com/index.affx”).Expression data were analyzed by the RMA (Robust Multichip Average)method in R using Bioconductor and associated packages (19), asdescribed (20, 21). RMA did background adjustment, quantilenormalization and summarization of 11 oligonucleotides per gene.

Before analysis, a filtering process removed from the dataset genes withlow and poorly measured expression as defined by expression valueinferior to 100 units in all the 16 samples, retaining 25,285genes/ESTs. A second filter, based on the intensity of standarddeviation (SD), was applied for unsupervised analyses to exclude genesshowing low expression variation across the analyses. SD was calculatedon log 2-transformed data, in which lowest values were first floored toa minimal value of 100 units, i.e. the background intensity, retaining13,550 genes/ESTs with SD superior to 0.5. An unsupervised analysis wasdone on 16 ALDEFLUOR-positive, -negative cells on 13,550 genes. Beforehierarchical clustering, filtered data were log 2-transformed andsubmitted to the Cluster program (22) using data median-centered ongenes, Pearson correlation as similarity metric and centroid linkageclustering. Results were displayed using TreeView program (22). Toidentify and rank genes discriminating ALDEFLUOR-positive and -negativepopulations, a Mann and Whitney U test was applied to the 25,285genes/ESTs and false discovery rate (FDR, (23) was used to correct themultiple testing hypothesis. The classification power of thediscriminator signature was illustrated by classifying samples byhierarchical clustering. A LOOCV was applied to estimate the accuracy ofprediction of the identified molecular signatures and the validity ofsupervised analysis; each sample was excluded one by one and classifiedwith the linear discriminant analysis (LDA, (24) by using model definedon the non-excluded samples.

Real-Time RT-PCR.

After ALDEFLUOR-positive and ALDEFLUOR-negative populations fromdifferent cell lines were sorted, total RNA was isolated using RNeasyMini Kit (QIAGEN) and utilized for real-time quantitative RT-PCR(qRT-PCR) assays in a ABI PRISM® 7900HT sequence detection system with384-well block module and automation accessory (Applied Biosystems).Primers and probes for the Taqman system were selected from the AppliedBiosystems website. The sequences of the PCR primer pairs andfluorogenic probes used for CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1 and TBPare available on the Applied Biosystems website (CXCR1 assay ID:Hs_00174146_mi; FBXO21 assay ID: Hs_00372141_mi, NFYA assay ID:Hs_00953589_mi, NOTCH2 assay ID: Hs_01050719_mi, RAD51L1 assay ID:Hs_00172522_mi, TBP assay ID: Hs_00427620_mi). The relative expressionmRNA level of CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1 was computed withrespect to the internal standard TBP gene to normalize for variations inthe quality of RNA and the amount of input cDNA, as described previously(25).

Invasion Assay.

Assays were done in triplicate in transwell chambers with Burn porepolycarbonate filter inserts for 12-well plates (Corning, N.Y.). Filterswere coated with 30 ul of ice-cold 1:6 basement membrane extract(Matrigel, BD-Bioscience) in DMEM/F12 incubated 1 hour at 37° C. Cellswere added to the upper chamber in 200 ul of serum-free medium. For theinvasion assay, 5000 cells were seeded on the Matrigel-coated filtersand the lower chamber was filled with 600 ul of medium supplemented with10% human serum (Cambrex) or with 600 ul of serum-free mediumsupplemented with IL8 (100 ng/mL). After 48 hours incubation, the cellson the underside of the filter were counted using light microscopy.Relative invasion was normalized to the unseparated corresponding celllines under serum condition.

Lentivirus Infection.

For luciferase gene transduction, 70% confluent cells from HCC1954,MDA-MB-453, and SUM159 were incubated overnight with a 1:3 precipitatedmixture of lentiviral supernatants Lenti-LUC-VSVG (Vector Core, AnnArbor, Mich.) in culture medium. The following day the cells wereharvested by trypsin/EDTA and subcultured at a ratio of 1:6. After 1week incubation, cells were sorted according to the ALDEFLUOR phenotypeand luciferase expression was verified in each sorted population(ALDEFLUOR-positive and ALDEFLUOR-negative) by adding 2 ml D-luciferin0.0003% (Promega, Madison, Wis.) in the culture medium and countingphoton flux by device camera system (Xenogen, Alameda, Calif.).

Intracardiac Inoculation.

Six weeks-old NOD/SCID mice were anesthetized with 2% isofluorane/airmixture and injected in the heart left ventricle with 100,000 cells in100 μL of sterile Dulbecco's PBS lacking Ca2+ and Mg2+. For each of thethree cell lines (HCC1954, MDA-MB-453, SUM159) and for each population(ALDEFLUOR-positive, ALDEFLUOR-negative and unsorted), three animalswere injected.

Bioluminescence Detection.

Baseline bioluminescence was assessed before inoculation and each weekthereafter inoculations. Mice were anesthetized with a 2%isofluorane/air mixture and given a single i.p. dose of 150 mg/kgD-luciferin (Promega, Madison, Wis.) in PBS. Animals were thenre-anesthetized 6 minutes after administration of D-luciferin. Forphoton flux counting, a charge-coupled device camera system (Xenogen,Alameda, Calif.) was used with a nose-cone isofluorane delivery systemand heated stage for maintaining body temperature. Results were analyzedafter 2 to 12 minutes of exposure using Living Image software providedwith the Xenogen imaging system. Signal intensity was quantified as thesum of all detected photon flux counts within a uniform region ofinterest manually placed during data postprocessing. Normalized photonflux represents the ratio of the photon flux detected each week afterinoculations and the photon flux detected before inoculation.

Statistical Analysis.

Results are presented as the mean±SD for at least three repeatedindividual experiments for each group. Statistical analyses used theSPSS software (version 10.0.5). Correlations between sample groups andmolecular parameters were calculated with the Fisher's exact test or theone-way ANOVA for independent samples. A p-value *0.05 was consideredsignificant.

The Majority of Breast Cell Lines Contain an ALDEFLUOR-PositivePopulation.

The ALDEFLUOR assay (17) was used to isolate CSC from 33 BCLsrepresenting the diverse molecular subtypes and features of breastcancer (20). It was found that 23 out of the 33 cell lines contained anALDEFLUOR-positive cell population that ranged from 0.2 to nearly 100%.All 16 basal/mesenchymal BCLs contained an ALDEFLUOR-positive populationwhereas 7 out of the 12 luminal BCLs did not contain any detectableALDEFLUOR-positive cells (p=0.0006, Fischer's exact test).

ALDEFLUOR-Positive Cells have Tumorsphere-Forming Capacity.

It has previously been reported that mammary epithelial stem andprogenitor cells are able to survive and proliferate inanchorage-independent conditions and form floating spherical colonieswhich are termed mammospheres (18). Data from breast tumors, as well ascell lines, have demonstrated that cancer stem-like cells orcancer-initiating cells can also be isolated and propagated as“tumorspheres” in similar assays (26). All mammosphere-initiating cellsin the normal human mammary gland are contained within theALDEFLUOR-positive population (17). To characterize theALDEFLUOR-positive population from BCLs, the ability ofALDEFLUOR-positive and -negative populations from 184A1, SUM149 andSUM159 to form tumorspheres were compared. In each cell line, theALDEFLUOR-positive population showed increased tumorsphere-formingcapacity compared to ALDEFLUOR-negative cells.

ALDEFLUOR-Positive BCL Cells have Cancer Stem Cell Properties In Vivo.

To determine the hierarchical organization of BCL, the stem cellproperties of the ALDEFLUOR-positive and -negative populations ofMDA-MB-453, SUM159, and BrCa-MZ-01 cell lines were analyzed. TheALDEFLUOR-positive populations of these three BCLs constituted between3.54±1.73% and 5.49±3.36% of the total cell populations (FIG. 1A-B, G-H;FIG. 2A-B). As shown in FIG. 1F, L the size and latency of tumorformation correlated with the number of ALDEFLUOR-positive cellsinjected. Remarkably, 500 ALDEFLUOR-positive cells from MDA-MB-453 and1,000 ALDEFLUOR-positive cells from SUM159 were able to form tumors. Thetumor-generating capacity was maintained through serial passagesdemonstrating the self-renewal capacity of these cells. In contrast,ALDEFLUOR-negative cells failed to generate tumors, although limitedgrowth was produced when 50,000 ALDEFLUOR-negative MDA-MB-453 cells wereinjected. H&E staining of the fat pad sections confirmed that tumorsformed by ALDEFLUOR-positive cells contained malignant cells whereasonly residual Matrigel, apoptotic cells and mouse tissue were seen atthe sites of ALDEFLUOR-negative cell injections (FIG. 1E, K). Consistentwith the ALDEFLUOR-positive population having cancer stem cellcharacteristics, tumors generated by this population recapitulated thephenotypic heterogeneity of the initial tumor, with a similar ratio ofALDEFLUOR-positive and -negative cells (FIG. 1C, I). This indicates thatALDEFLUOR-positive cells were able to self-renew, generatingALDEFLUOR-positive cells and were able to differentiate, generatingALDEFLUOR-negative cells.

When BrCa-MZ-01 cells were separated into ALDEFLUOR-positive and-negative components, both were capable of tumor generation. Tumorsgenerated by the ALDEFLUOR-positive population consisted of bothALDEFLUOR-positive and -negative cells recapitulating the phenotypicheterogeneity of the initial tumor. In contrast, tumors generated byALDEFLUOR-negative cells gave rise to slowly growing tumors containingonly ALDEFLUOR-negative cells. In contrast to the ability ofALDEFLUOR-positive cells to be serially transplanted, serial passages ofALDEFLUOR-negative tumors produced decreasing tumor growth with nogrowth following three passages. This suggests that theALDEFLUOR-positive component of the BrCa-MZ-01 cells contain cells withstem cell properties, whereas the ALDEFLUOR-negative cells containprogenitor cells able to undergo limited growth but not self-renewal.

Gene Expression Profiling of ALDEFLUOR-Positive and -Negative CellPopulations.

To determine whether ALDEFLUOR-positive cells isolated from differentBCLs expressed a common set of “cancer stem cell” genes, theALDEFLUOR-positive and -negative cell populations isolated from eightBCLs (184A1, BrCa-MZ-01, HCC1954, MDA-MB-231, MDA-MB-453, SK-BR-7,SUM49, and SUM159) were analyzed using Affymetrix whole-genomeoligonucleotide microarrays. Unsupervised hierarchical clustering,applied to the 16 samples and the 13,550 filtered genes/ESTs, did notseparate ALDEFLUOR-positive and -negative populations. Instead,ALDEFLUOR-positive and -negative populations clustered with the parentalcell line. This suggests that the differences in mRNA transcriptsbetween clonal cell lines supersede differences betweenALDEFLUOR-positive and ALDEFLUOR-negative cells. This further suggeststhat only a limited number of genes are differentially expressed betweenputative cancer stem cells and their progeny.

To determine which genes discriminated ALDEFLUOR-positive and -negativepopulations, the Mann and Whitney U test was applied to all genes butthose with low and poorly measured expression, i.e. 25,285 probe sets.This test identified and ranked after FDR correction, 413 genes/ESTsthat discriminated the ALDEFLUOR-positive and -negative cellpopulations. The 28 overexpressed genes corresponding to unique genesare shown in Table 1, and the most frequently underexpressed genes areshown in Table 2.

TABLE 1 Up Regulated Genes Category Symbol Description Cytoband Probeset ID Function Genes TPRXL tetra-peptide repeat homeobox-like chr3p25.1239061_at early embryonic development previously described to have arole in stem cell biology NOTCH2 Notch homolog 2 (Drosophila)chr1p13-p11 202443_x_at Self-renewal program RBM15 RNA binding motifprotein 15 chr1p13 1555760_a_at determination of hematopoietic cell fateST3GAL3 ST3 beta-galactoside alpha-2,3- chr1p34.1 1555181_a_atmaintenance of the embryonic sialyltransferase 3 antigens SSEA-3 and -4NFYA nuclear transcription factor Y, alpha chr6p21.3 204107_atSelf-renewal program PCNX pecanex homolog (Drosophila) chr14q24.2213173_at determination of neural cell fate of early developing embryoSignaling FBXO21 * F-box protein 21 chr12q24.22 212231_at UbiquitinationWWOX WW domain containing oxidoreductase chr16q23.3-q24.1 210695_s_atProtein degradation, transcription, and RNA splicing CAMK2BCalcium/calmodulin-dependent protein kinase chr22q12 34846_at Calciumsignaling (CaM kinase) II beta PNPLA2 patatin-like phospholipase domaincontaining 2 chr11p15.5 39854_r_at Triglyceride hydrolysis CLIC5chloride intracellular channel 5 chr6p12.1-21.1 213317_at chloride iontransport UGCGL1 UDP-glucose ceramide glucosyltransferase-like 1chr2q14.3 222569_at Protein glucosylation FBXL18 F-box and leucine-richrepeat protein 18 chr7p22.2 220896_at Ubiquitination ADRBK1 adrenergic,beta, receptor kinase 1 chr11q13 38447_at Phosphorylation of G-protein-coupled receptors SLC38A2 Solute carrier family 38, member 2 chr12q1559924_at neutral amino acid transporter Membrane IL8RA* interleukin 8receptor, alpha chr2q35 207094_at Inflamatory response protein (CXCR1)TAS2R14 Taste receptor, type 2, member 14 chr12p13 241997_at Bitterperception CD300LB CD300 molecule-like family member b chr17q25.11554173_at Immune response GIPC3 GIPC PDZ domain containing family,member 3 chr19p13.3 236730_at DNA repair RAD51L1 RAD51-like 1 (S.cerevisiae) chr14q23-q24.2 1570166_a_at homologous recombination repairChromatin ARID1B AT rich interactive domain 1B (SWI1-like) chr6q25.1225181_at Chromatin remodeling (SWI/SNF remodeling complexe)Cytoskeleton EPPK1 epiplakin 1 chr8q24.3 208156_x_at Maintenance of thekeratin intermediate filaments Extracellular COL11A2 collagen, type XI,alpha 2 chr6p21.3 216993_s_at skeletal morphogenesis matrix KLK3Kallikrein 3, (prostate specific antigen) chr19q13.41 231629_x_atProtease RNA EIF2C2 Eukaryotic translation initiation factor 2C, 2chr8q24 213310_at short-interfering-RNA-mediated interference genesilencing Unknown ZFP41 zinc finger protein 41 homolog (mouse) chr8q24.3227898_s_at Unknown FAM49B Family with sequence similarity 49, member Bchr8q24.21 243182_at Unknown PSORS1C2 psoriasis susceptibility 1candidate 2 chr6p21.3 220635_at Unknown

TABLE 2 Down Regulated Genes Category Symbol Description Cytoband Probeset ID Function Protein MRPL42* mitochondrial ribosomal protein L42chr12q22 217919_s_at Protein synthesis synthesis within themitochondrion MRPL54* mitochondrial ribosomal protein L54 chr19p13.3225797_at Protein synthesis within the mitochondrion MRPL47*mitochondrial ribosomal protein L47 chr3q26.33 223480_s_at Proteinsynthesis within the mitochondrion MRPS23* mitochondrial ribosomalprotein S23 chr17q22-q23 223156_at Protein synthesis within themitochondrion EIF3S9* eukaryotic translation initiation factor 3,chr7p22.2 236274_at Initiation of subunit 9 eta, 116 kDa proteinsynthesis (EIF3 multiprotein complex) Signaling ALG5* asparagine-linkedglycosylation 5 homolog chr13q13.3 218203_at Protein glucosylationDNAJC19* DnaJ (Hsp40) homolog, subfamily C, chr3q26.33 225359_atImportation of member 19 mithochondrial protein HBLD2* HESB like domaincontaining 2 chr9q21.33 221425_s_at iron-sulfur cluster biogenesis GART*Phosphoribosylglycinamide chr21q22.1 230097_at de novo purineformyltransferase, biosynthesis phosphoribosylglycinamide synthetase,phosphoribosylaminoimidazole synthetase NUP37* nucleoporin 37 kDachr12q23.2 218622_at Intrcellular protein transport across nuclearmembrane RNF7* ring finger protein 7 chr3q22-q24 224439_x_at subunit ofSKP1-cullin/ CDC53-F box protein ubiquitin ligases DC2* DC2 proteinchr4q25 223001_at protein glycosylation USP15* ubiquitin specificpeptidase 15 chr12q14 210681_s_at Protein degradation COMMD6* COMMdomain containing 6 — 225312_at Inhibition NF-KappaB Signaling UBL5*Ubiquitin-like 5 chr19p13.3 218011_at Ubiquitination Apoptosis MRPL41*mitochondrial ribosomal protein L41 chr9q34.3 225425_s_at Stabilizationof p53 protein, Cell cycle arrest (p21(WAF1/CIP1) and p27(Kip1)dependent) PDCD10* programmed cell death 10 chr3q26.1 210907_s_atInitiation of apoptosis PDCD5* Programmed cell death 5 chr19q12-227751_at Initiation of apoptosis Differentiation NACA*nascent-polypeptide-associated complex chr12q23-q24.1 222018_atErythroid program alpha polypeptide differentiation Cell cycle FAM82B*family with sequence similarity 82, member B chr8q21.3 218549_s_atRegulation of microtubule dynamic RNA splicing CCNL1* cyclin L1chr3q25.32 1555411_a_at Pre-mRNA processing PRPF39* PRP39 pre-mRNAprocessing factor 39 chr14q21.3 220553_s_at Pre-mRNA homolog (S.cerevisiae) processing LSM3* LSM3 homolog, U6 small nuclear RNAchr3p25.1 202209_at Pre-mRNA associated (S. cerevisiae) processingSFRS7* splicing factor, arginine/serine-rich 7, chr2p22.1 213649_atRegulation of 35 kDa RNA splicing PRPF4B* PRP4 pre-mRNA processingfactor 4 chr6p25.2 202127_at Pre-mRNA processing homolog B (yeast)Oxidative ATP5S* ATP synthase, H+ transporting, chr14q22.1 206992_s_atsubunit of phosphorylation mitochondrial F0 complex, subunit s (factormitochondrial B) ATP synthase NDUFA2* NADH dehydrogenase (ubiquinone) 1chr5q31 209224_s_at components of alpha subcomplex, 2, the complex Imulti-subunit enzyme ATP5J2* ATP synthase, H+ transporting, chr7q22.1202961_s_at subunit of mitochondrial F0 complex, subunit F2mitochondrial ATP synthase IMMP1L* IMP1 inner mitochondrial membranechr11p13 230556_at Proteolysis peptidase-like Unknown ASTE1* asteroidhomolog 1 (Drosophila) chr3q22.1 221135_s_at Unknown MGC61571*hypothetical protein MGC61571 chr3p24.1 228283_at Unknown WDR53* WDrepeat domain 53 chr3q29 227814_at Unknown DKFZP686A10121* hypotheticalprotein chr7q21.13 234311_s_at Unknown CHCHD8*coiled-coil-helix-coiled-coil-helix domain chr11q13.4 220647_s_atUnknown containing 8 FLJ32745* hypothetical protein FLJ32745 chr2q13235644_at Unknown CHURC1* churchill domain containing 1 chr14q23.3233268_s_at Unknown XTP3TPA* XTP3-transactivated protein A chr16p11.2218069_at Unknown FLJ37953* hypothetical protein FLJ37953 chr2q33.1235181_at Unknown SNORD50A* small nucleolar RNA, C/D box 50A chr6q14.3244669_at Unknown LOC644053* hypothetical protein LOC644053 chr1q41235466_s_at Unknown TMEM141* transmembrane protein 141 chr9q34.3225568_at Unknown C8orf59* chromosome 8 open reading frame 59 chr8q21.2226165_at Unknown

The classification power of this discriminating signature wasillustrated by classifying the 16 ALDEFLUOR-positive and -negativesamples with the 413 differentially expressed genes/ESTs. Hierarchicalclustering ranked 15 out of the 16 samples (FIG. 2A).

A number of genes known to play a role in stem cell biology wereupregulated in the ALDEFLUOR-positive populations (Table 1), includingNFYA, NOTCH2, PCNX, RBM15, ST3GAL3, and TPRXL. Other genes encodeproteins that have putative or uncharacterized role in stem cellfunction, such as ARID1B, RAD51L1, and the chemokine receptorCXCR1/IL8RA (27). Genes underexpressed in the ALDEFLUOR-positivepopulation are involved in cell differentiation, apoptosis, RNAsplicing, and mitochondrial metabolism.

To increase the stringency of analysis, the threshold of the Mann andWhitney analysis was raised to the 0.5 risk and obtained a list of 49genes/ESTs that discriminated ALDEFLUOR-positive and -negativepopulations (genes with asterisk in Tables 1-2). With this list, all ofthe ALDEFLUOR-positive cells, except from SK-BR-7, clustered together.Among these 49 genes/ESTS, 45 corresponded to identified unique genes;only 3 of these 45 were overexpressed in the ALDEFLUOR-positive groupwhile 42 were underexpressed. Characterized overexpressed genes code foran F-box protein FBXO21 and CXCR1/IL8RA. Underexpressed genes includethose coding for mitochondrial proteins (MRPL41, MRPL42, MRPL47, MRPL54,MRPS23, IMMP1L), and differentiation (NACA) and pre-mRNA splicingfactors (LSM3, pre-mRNA processing factor PRPF39 and PRPF4B).Leave-one-out cross-validation (LOOCV) at 0.5% risk estimated theaccuracy of prediction of the identifier molecular signature and 88% ofthe samples were predicted in the right class with this “cancer stemcell signature” confirming the supervised analysis.

Quantitative RT-PCR assessment confirmed a significant increase of CXCR1and FBXO21 in ALDEFLUOR-positive cells. Quantitative RT-PCR analysis offive discriminator genes overexpressed in ALDEFLUOR-positive populations(CXCR1/IL8RA, FBXO21, NFYA, NOTCH2 and RAD51L1) was performed. Threecell lines used in the profiling analysis (BrCa-MZ-01, MDA-MB-453,SUM159) and two additional luminal cell lines (MCF7, S68) were sorted byALDEFLUOR-assay and ALDEFLUOR-positive and -negative populations wereprocessed separately for quantitative RT-PCR analysis. The quantitativeRT-PCR expression level of CXCR1 and FBXO21 are presented in FIGS. 2 Band C. Gene expression levels measured by quantitative RT-PCR confirmedthe results obtained using DNA microarrays with an increase of CXCR1 andFBXO21 mRNA level in the ALDEFLUOR-positive population compared to theALDEFLUOR-negative population (p<0.05).

IL8 Promotes Cancer Stem Cell Self-Renewal.

The profiling studies suggested that the IL8 receptor CXCR1/IL8RA wasconsistently expressed in the ALDEFLUOR-positive cell population. Toconfirm this association, the protein expression of CXCR1/IL8RA wasmeasured by flow cytometry in ALDEFLUOR-positive and -negativepopulations. The ALDEFLUOR-positive and -negative populations from fourdifferent cell lines were isolated by FACS, fixed, and stained with aCXCR1 monoclonal antibody labeled with phycoerythrin. As shown in FIG.3A, ALDEFLUOR-positive cells were highly enriched in CXCR1-positivecells compared to the ALDEFLUOR-negative populations.

To determine whether IL8 signaling is important in stem cell function,four BCLs were treated with human recombinant IL8 to determine itseffect on the cancer stem cell population as measured by the formationof tumorspheres and by ALDH enzymatic activity. As shown in FIG. 3B,addition of IL8 increased the formation of primary and secondarytumorspheres in a dose-dependent manner. Furthermore, IL8 increased theALDEFLUOR-positive population in a dose-dependent manner in each of thefour BCLs analyzed (FIG. 3C). This illustrates the power of the “CSCsignature” to identify pathways that may play a role in stem cellfunction.

The IL8/CXCR1 Axis is Involved in Cancer Stem Cell Invasion.

The IL8/CXCR1 axis has been reported to play a role in cancer stem cellinvasion (28, 29). A Matrigel invasion assay was utilized, using serumas attractant, to examine the ability of ALDEFLUOR-positive and-negative cell populations from three different cell lines (HCC1954,MDA-MB-453, SUM159) to invade. As shown in FIG. 4A, ALDEFLUOR-positivecells demonstrated 6- to 20-fold higher invasion through Matrigel thanthe ALDEFLUOR-negative population (p<0.01). When used as achemo-attractant IL8 (100 ng/ml) increased invasion of theALDEFLUOR-positive cells (p<0.05) (FIG. 4A). In contrast to its effectson ALDEFLUOR-positive cells, IL8 did not have any effect on the invasivecapacity of ALDEFLUOR-negative cells. These results indicate that cancerstem cells exhibited invasive behavior and furthermore that IL8facilitates this process.

ALDEFLUOR-Positive Cells have Increased Metastatic Potential.

It has been proposed that CSCs play a crucial role in cancer metastasis(30, 31). The above experiments demonstrated that ALDEFLUOR-positivecells have increased invasive capacity compared to ALDEFLUOR-negativecells. To determine the relationship between ALDEFLUOR-positivity andmetastatic capacity, HCC1954, MDA-MB-453, and SUM159 were infected witha luciferase lentivirus reporter system. Luciferase-infected cells weresorted using the ALDEFLUOR assay and introduced into NOD/SCID mice byintracardiac injection. A suspension of 100,000 cells from eachpopulation was injected and metastasis was assessed by bioluminescentimaging. Mice inoculated with ALDEFLUOR-positive cells developedmetastases at different sites and displayed a higher photon fluxemission than mice inoculated with unseparated cells, which developed nomore than one metastasis per mouse, or mice inoculated withALDEFLUOR-negative cells, which developed only occasional metastaseslimited to lymph nodes (FIG. 4B-J). Histologic sections confirmed thepresence of metastases at these sites (FIG. 4K-M). Thus, the metastaticcapacity of BCLs is predominantly mediated by CSCs contained in theALDEFLUOR-positive population.

The hypothesis that tumors are organized in a cellular hierarchy drivenby CSCs has fundamental implications for cancer biology as well asclinical implications for the early detection, prevention and treatmentof cancer. Evidence for CSCs has largely relied on primary and earlypassage xenograft models (32-34). However, the success of establishingbreast tumor xenograft has been low particularly for certain molecularsubtypes. In contrast to primary tumors, cell lines are available inunlimited quantities and provide only carcinomatous populations formolecular analysis without normal tissue and stroma. In breast cancer, alarge number of immortalized cell lines have been produced whichrepresent the different molecular subtypes found in primary human breastcancers (2, 20). However, a fundamental question remains as to howclosely these cell lines are able to recapitulate the biology of humanbreast cancer.

In Vivo Evidence for Stem Cells in Cell Lines.

Recent studies have suggested that although cell lines may be clonallyderived, they contain a cellular hierarchy representing different stagesof cellular differentiation. Several studies have utilized markers suchas CD44+/CD24− to identify CSC within breast cancer cell lines. However,their utility is limited by the observation that frequently a largepercentage of cells within a cell line express these putative stem cellmarkers. For example, greater than 90% of cells in basal breast cancercell lines display the CD44+/CD24− phenotype. Indeed, theCD44+/CD24-phenotype did not isolate the tumorigenic population of thesecell lines (Ginestier et al. Cell Stem Cell 1:555-567., hereinincorporated by reference in its entirety). An alternative approach hasbeen to use the SP from cell lines. However, functional studiesutilizing Hoechst staining are limited by the toxicity of this agent(35). There is also evidence that the functional stem cell activity isnot contained within the SP(36).

ALDH activity assessed by the ALDEFLUOR assay isolates cells with stemcell properties from various cancers (14, 37). In this Example it wasdemonstrated that 23 out of 33 BCLs (predominantly basal cell lines)contain an ALDEFLUOR-positive population. Lack of an ALDEFLUOR-positivepopulation in some luminal BCLs may indicate that these luminal BCLs arederived from ALDEFLUOR-negative progenitor cells.

This Example utilized in vivo assays in NOD/SCID mice to demonstrate thestem cell properties of the ALDEFLUOR-positive populations. Self-renewalwas demonstrated by serial passage in NOD/SCID mice and differentiationwas demonstrated by the ability of ALDEFLUOR-positive but notALDEFLUOR-negative cells to regenerate the cellular heterogeneity of theinitial tumor.

A Breast Cancer Stem Cell Signature.

Utilizing eight breast cell lines, this Example identified 413 geneswhose expression discriminates ALDEFLUOR-positive and -negative cells.This signature contained a number of genes known to play a role in stemcell biology. Genes overexpressed in the ALDEFLUOR-positive populationinclude Notch homolog 2 (NOTCH2), which regulates self-renewal anddifferentiation of mammary stem cells (18, 38), NFYA, known to regulateself-renewal and differentiation of stem cells. (39, 40), pecanexhomolog PCNX, RBM15/OTT, which plays a pleiotropic role in hematopoieticstem cells (41) and affects myeloid differentiation via NOTCH signaling(42), homeobox-like factor TPRXL involved in embryonic development,ST3GAL3, which codes for a stage-specific embryonic antigen-4 synthase,associated with fetal development and renal and gastric carcinogenesis(43). Notably, stage-specific embryonic antigen-4 protein (SSEA-4) isexpressed in stem cell populations such as CXCR4+/CD133+/CD34+/lin− stemcells in human cord blood and quiescent mammary stem cells (44).

Genes underexpressed in the ALDEFLUOR-positive population are involvedin cell differentiation, apoptosis, and mitochondrial oxidation. Theyinclude genes coding for nascent polypeptide-associated complex alphasubunit NACA, programmed death proteins PDCD5 and PDCD10, mitochondrialribosomal protein L41 (MRPL41), which induces apoptosis throughP53-dependent and independent manner via BCL2 and caspases, and proteinsinvolved in mitochondrial processes such as oxidative phosphorylation(NDUFA2, ATP5J2, IMMP1L) and protein synthesis in the mitochondrion(MRPL42, MRPL47, MRPL54, MRPS23). Downregulation of apoptotic genes inCSCs may play a role in the resistance of these cells to radiation andchemotherapy (45, 46). ALDH1A1 was not identified as adifferentially-expressed gene in the ALDEFLUOR-positive signature.However, examination of gene expression profile of individual BCLsrevealed that although some showed differential expression of ALDH1A1 inthe ALDEFLUOR-positive population, others showed differential expressionof ALDH1A3, a different ALDH isoform in this population. This suggeststhat the expression of different ALDH isoforms could contribute to theALDEFLUOR-positive phenotype.

From Chemokines to “Stemokines.”

The expression of CXCR1, a receptor for IL8, is increased in a varietyof cancers (47-50). Although IL8 expression is associated withER-negative breast cancer (51), this chemokine has not previously beenreported to play a role in stem cell function. Its implication in theregulation of growth and metastasis is well-established inandrogen-independent prostate cancer (52). Furthermore, the expressionlevel of IL8 is associated with tumorigenicity and metastasis throughVEGF production and angiogenesis (53, 54). The gene expression data wasvalidated in three ways. First, quantitative RT-PCR analysis confirmed asignificant increase of CXCR1 mRNA in ALDEFLUOR-positive population fromcell lines both included and not included in profiling analysis. Second,it was demonstrated using flow cytometry that CXCR1-containing cellswere found exclusively within the ALDEFLUOR-positive population. Third,recombinant IL8 increased mammosphere formation and the percent ofALDEFLUOR-positive cells in BCLs. The IL8/CXCR1 axis thus appears toregulate mammary stem cell proliferation or self-renewal. Sinceendothelial and stromal cells secrete IL8 this chemokine appears to playa role in mediating interactions between tumor stem cells and the tumormicroenvironment.

Recent studies have suggested a role for interleukines/chemokines in theregulation of CSCs (55, 56). This includes a role for IL6 in breast CSCsand IL4 in mediating chemoresistance of colon CSCs (56-59). Thesefactors may be involved in the association between inflammation andcancer. This also includes a role for CCL5 (RANTES), a chemokinesecreted by mesenchymal stem cells, which acts as a paracrine factor andenhance breast cancer cells motility, invasion and metastasis(55).

The Roots of Metastasis.

CSCs may be responsible for mediating tumor metastasis. A link betweenCSC and metastasis was first suggested with the identification of stemcell genes in an 11-gene signature generated using comparative profileof metastastatic and primary tumors in transgenic mouse model ofprostate cancer and cancer patients (60). This signature was also apowerful predictor of disease recurrence, death after therapy anddistant metastasis in a variety of cancer types. This Example hasdemonstrated that ALDEFLUOR-positive cells are more metastatic thanALDEFLUOR-negative cells and that IL8, previously reported to play arole in tumor metastasis, promotes the invasion and chemotaxis of cancerstem cells which preferentially express the IL8 receptor CXCR1. Theability to isolate metastatic cancer stem cell from cell lines shouldfacilitate studies of the molecular mechanisms by which cancer stemcells mediate tumor metastasis.

Example 2 CXCR1 Inhibition and Combination Therapy

This example describes various methods employed to test the effect ofCXCR1 inhibition on tumor cells, as well as the combination of CXCR1inhibition in combination with an anti-mitotic agent (docetaxel).

Effect of CXCR1 Inhibition on the Cell Growth and on the ALDEFLUORPositive Population of SUM159 Cell Line.

The SUM159 cell line was cultured in adherent condition and treated thecells using the CXCR1/CXCR2 inhibitor Repertaxin or two specificblocking antibodies for CXCR1 or CXCR2. After 4 days of treatment, theeffect on cell growth was analyzed using the MTT assay (FIG. 5A) and onthe cancer stem cell population using the ALDEFLUOR assay (FIG. 5B).More than 95% of cell growth inhibition was observed in the cellstreated with Repertaxin or the CXCR1 blocking antibody, whereas noeffect was observed for the cells treated with the CXCR2 blockingantibody (FIG. 5A). Interestingly similar effect was observed on theALDEFLUOR-positive population with a decrease of 80% and 50% of theALDEFLUOR-positive population in the cells treated with Repertaxin andCXCR1 blocking antibody respectively (FIG. 5B).

Repertaxin Treatment Induces a Bystander Effect Mediated by the FAS/FASLigand Signaling

SUM159 cell line cells were cultured in adherent conditions and thentreated with Repertaxin alone or in combination with a FAS antagonist.Interestingly, the cell growth inhibition induced by the Repertaxintreatment was partially rescued by the addition of a FAS antagonist(anti/Fas-ligand from BD pharmingen (cat#556371)). Moreover, the cellstreated with a FAS agonist displayed a similar cell growth inhibitionthan the cells treated with Repertaxin. These results suggest thatRepertaxin treatment induces a bystander effect mediated by the FAS/FASligand signaling.

Effect of Repertaxin Treatment on FAK, AKT and FOXOA3 Activation.

In order to evaluate the effect of Repertaxin treatment on the CXCR1downstream signaling, SUM159 cells were cultured, during 2 days, inadherent condition in the absence or in presence of 100 nM of Repertaxinand stained by immunofluorescence with antibodies against p-FAK, p-AKT,and FOXOA3. In the non-treated cells (FIG. 7A), it was detected that 30%of cells expressing p-FAK and 10% of cells expressing p-AKT displayedinactivation, while cells treated with Repertaxin displayed a completeinactivation of p-FAK and p-AKT (FIG. 7B). The non-treated SUM159 cellspresented 80% of cells positive in the cytoplasm for FOXOA3.Interestingly, SUM159 cells treated with Repertaxin presented 80% ofcells positive in the nucleus for FOXOA3. The change in FOXOA3 cellularlocation from the cytoplasm to the nucleus indicates an activation ofFOXOA3 protein.

Tumors Growth Curves Following the Treatment with Repertaxin, Docetaxelor the Combination

The effect of Repertaxin, docetaxel, or the combination thereof wasevaluated using one breast cancer cell lines (8A, SUM159) and threehuman breast cancer xenografts generated from different patients (8B,MC1; 8C, UM2; and 8D, UM3). For each sample, 50,000 cells were injectedinto the mammary fat pad of NOD-SCID mice which were monitored for tumorsize. Injections were started when the tumor size was about 4 mm.Repertaxin was injected (15 mg/Kg) twice a day for 28 days or once aweek, docetaxel was I.P. injected (10 mg/Kg), or the combination(Repertaxin/Docetaxel) was employed. FIG. 8 shows the tumor sizes beforeand during the course of each indicated treatment (arrow, beginning ofthe treatment). Similar results are observed for each sample (SUM159,MC1, UM2, UM3) with a statistically significant reduction of the tumorsize when treated with Docetaxel alone or the combinationRepertaxin/Docetaxel compared to the control (p<0.01) whereas nosignificant difference are observed between the growth of the controltumors and the tumors treated with Reperataxin.

Effect of Repertaxin, Docetaxel, or the Combination Treatment on theCancer Stem Cell Population as Assessed by the ALDEFLUOR Assay

ALDH activity was assessed by the ALDEFLUOR assay for analyzing thecancer stem cell populations size in each tumor (9A. SUM159, 9B. MC1,9C. UM2, 9D. UM3) treated with Repertaxin, docetaxel or the combination.Similar results are observed for each sample. Docetaxel treated tumorxenografts showed similar or increase percentage of ALDEFLUOR-positivecells compare to the control, whereas Repertaxin treatment alone or incombination with docetaxel produced a statistically significant decreasein ALDEFLUOR-positive cells with 65% to 85% less cancer stem cellscompare to the control (p<0.01).

Effect of Repertaxin, Docetaxel, or the Combination Treatment on theCancer Stem Cell Population as Assessed by Implantation in SecondaryMice.

Serial dilutions of cells obtained from primary tumors (10A. SUM159,10B. MC1, 10C. UM2, 10D. UM3) non treated (control) and treated withRepertaxin, docetaxel or the combination were implanted in the mammaryfat pad of secondary NOD-SCID mice. Control and docetaxel treatedprimary tumors formed secondary tumors at all dilutions whereas, onlyhigher concentration of primary tumors treated with Repertaxin or incombination with docetaxel were able form delayed secondary tumors whichwere significantly smaller in size than the control or docetaxel treatedtumors (p<0.01). Moreover, 1000 and 100 primary treated cells with thecombination failed to form secondary tumors for 3 out of 4 samples(SUM159, UM2, UM3).

Repertaxin Treatment Reduces the Metastatic Potential of SUM159 CellLine

A SUM159 cell line was infected with a lentivirus expressing luciferaseand inoculated 250,000 luciferase infected cells in the heart ofNOD/SCID mice. The mice were organized into two groups. The two groupsof mice were treated 12 hours after the intracardiac injection eitherwith s.c. injection of saline solution or s.c. injection of Repertaxin(15 mg/kg), twice a day during 28 days. Metastasis formation wasmonitored using bioluminescence imaging (11B: Mice treated with salinesolution; 11C: Mice treated with Repertaxin). Quantification of thenormalized photon flux measured at weekly intervals followinginoculation revealed a statistically significant increase of metastasisformation in the group of mice treated with saline solution compare tothe group of mice treated with Repertaxin (11A).

Example 3 Treatment of Cancer Stem Cells by CXCR1 Blockade

This example demonstrates the effect of CXCR1 inhibition on tumor cells,through both in vitro assays and mouse models.

Dissociation of Mammary Tissue.

100-200 g of normal breast tissue from reduction mammoplasties wasminced with scalpels, dissociated enzymatically, and single cells werecultured in suspension to generate mammospheres or on a collagensubstratum in adherent condition to induce cellular differentiation(Dontu et al. Genes Dev. 17:1253-1270, herein incorporated by referencein its entirety).

Cell Culture.

Breast cancer cell lines were grown using recommended culture conditions(Charafe-Jauffret et al. Cancer Res. 69:1302-1313., herein incorporatedby reference in its entirety). Breast cancer cell lines were treated inadherent condition with repertaxin (Sigma-Aldrich), anti-human CXCR1mouse monoclonal antibody (Clone 42705, R&D systems), anti-human CXCR2mouse monoclonal antibody (clone 48311, R&D systems), anti-human CD95mouse monoclonal antibody (Clone DX2, BD Pharmingen) utilized as a FASsignaling agonist, anti-human FAS-Ligand mouse monoclonal antibody(Clone NOK-1, BD pharmingen) utilized as a FAS signaling antagonist, orwith docetaxel (Taxotere, Sanofi-Aventis).

Cell Viability.

For MTT assays, cells were plated in adherent condition in 96-wellplates at 5,000 cells per well. After one day, treatment with repertaxinwas started. The effect of repertaxin treatment on cell viability wasestimated at different time points by addition of 20 μl of MTT solution(5 mg/mL in PBS) in each well. Cells were then incubated for 1 hour at37° C. followed by addition of 50 μL of DMSO to each well. Absorbancewas measured at 560 nm in a fluorescence plate reader (Spectrafluor,Tecan). For TUNEL assays, cells were plated in adherent conditions in6-well plates at 50,000 cells per well. After one day, treatment withrepertaxin was started. The number of apoptotic cells was estimatedafter four days treatment. Cells were fixed in 3.7% formaldehyde andstained utilizing the TACS TdT kit (R&D systems). Nuclei werecounterstained with DAPI/antifade (Invitrogen). Sections were examinedwith a fluorescent microscope (Leica, Bannockborn, Ill., USA) withapoptotic cells detected in green.

ALDEFLUOR Assay.

The ALDEFLUOR kit (StemCell technologies) was used to isolate thepopulation with high ALDH enzymatic activity using a FACStarPLUS (BectonDickinson) as previously described (Ginestier et al. Cell Stem Cell1:555-567., herein incorporated by reference in its entirety). In orderto eliminate cells of mouse origin from the xenotransplanted tumors,cell population was stained with an anti-H2Kd antibody (BD biosciences,1/200, 20 min on ice) followed by staining with a secondary antibodylabeled with phycoerythrin (PE) (Jackson labs, 1/250, 20 min on ice).

ELISA Assay.

To measure the level of soluble FAS-ligand secreted in the culturemedium of cells treated or not with repertaxin, Human sFAS Ligand Elisa(Bender Medsystems) was utilized. Absorbance was read on aspectro-photometer using 450 nm as the primary wave length.

Western Blotting.

Cells were lysed in a laemmli buffer and loaded onto SDS-polyacrylamidegels. Blots were incubated with the respective primary antibodiesdiluted in TBST (containing 0.1% Tween20 and 2% BSA) either overnight at4°, or 2 hours at room temperature. Blots were washed and incubated withappropriate secondary antibodies (GE Healthcare, UK) and detected usingSuperSignal West Pico Chemiluminescent Substrate (Pierce).

Immunostaining.

For immunofluorescent staining, sorted CXCR1-positive cells were fixedwith 95% methanol at −20° C. for 10 minutes. Cells were rehydrated inPBS and incubated with respective antibodies at room temperature for 1hour. Primary antibodies used were P-FAK (1:50, Cell SignalingTechnology), P-AKT (1:300, Cell Signaling Technology), and FOXO3a(1:250, Cell Signaling Technology). Slides were then washed andincubated 30 minutes with PE conjugated secondary antibodies (Jacksonlabs). The nuclei were counterstained with DAPI/antifade (Invitrogen)and coverslipped. Sections were examined with a fluorescent microscope(Leica, Bannockborn, Ill., USA). Immunohistochemistry for the detectionof ALDH1 (1:100, BD biosciences), P-FAK, P-AKT, FOXO3a expression wasdone on paraffin section (Ginestier et al. Am. J Pathol. 161:1223-1233.,herein incorporated by reference in its entirety). Staining was doneutilizing the Histostainplus kit (Zymed laboratories). Diaminobenzidine(DAB) or 3-amino-9-ethylcarbazole (AEC) was used as chromogen andsections were counterstained with hematoxylin.

Animal Model.

Tumorigenicity of ALDEFLUOR-positive/CXCR1-positive andALDEFLUOR-positive/CXCR1-negative SUM159 cells was assessed in NOD/SCIDmice (Ginestier et al. Cell Stem Cell 1:555-567., herein incorporated byreference in its entirety)., The SUM159 cell line and three primaryhuman breast cancer xenografts generated from three different patients(MC1, UM2, UM3) were utilized to determine the efficiency of repertaxintreatment on tumor growth (Ginestier et al. Cell Stem Cell 1:555-567.,herein incorporated by reference in its entirety). Cells from thesetumors were transplanted orthotopically in the humanized cleared fat-padof NOD/SCID mice, without cultivation in vitro. Fat pads were preparedas described previously (Ginestier et al. Cell Stem Cell 1:555-567.,herein incorporated by reference in its entirety). 50,000 cells fromeach xenotransplants were injected in the humanized fat pad of NOD/SCIDmice and monitored the tumor growth. When the tumor size wasapproximately 4 mm, treatment with repertaxin alone (s.c., 15 mg/Kg,twice a day, during 28 days), docetaxel alone (i.p., 10 mg/Kg, once aweek, during 4 weeks), in combination (repertaxin/docetaxel), or acontrol group injected with saline (i.p., once a week and s.c. twice aday, during 28 days) was initiated. The animals were euthanized when thetumors were approximately 1.5 cm in the largest diameter, to avoid tumornecrosis and in compliance with regulations for use of vertebrate animalin research. A portion of each fat pad injected was fixed in formalinand embedded in paraffin for histological analysis. The rest of thetumor cells were re-implanted into secondary NOD/SCID mice. Serialdilutions of cells were utilized for the re-implantation with injectionof 10,000, 1,000, and 100 cells for each treated tumor.

Anchorage-Independent Culture.

BCLs treated, in adherent conditions, with repertaxin (100 nM),anti-CXCR1 antibody (10 μg/ml), or anti-CXCR2 (10 μg/ml) weredissociated and plated as single cells in ultra-low attachment plates(Corning, Acton, Mass.) at low density (5,000 viable cells/ml). Cellswere grown as previously described (Charafe-Jauffret et al. Cancer Res.69:1302-1313., herein incorporated by reference in its entirety).Subsequent cultures after dissociation of primary tumorospheres wereplated on ultra-low attachment plates at a density of 5,000 viablecells/ml. The capacity of cells to form tumorspheres was quantifiedafter the first (primary tumorospheres) and second (secondarytumorospheres) passage.

RNA Extraction and qRT-PCR.

After SUM159 cells were treated, total RNA was isolated using RNeasyMini Kit (QIAGEN) and utilized for real-time quantitative RT-PCR(qRT-PCR) assays in a ABI PRISM® 7900HT sequence detection system.Primers and probes for the Taqman system were selected from the AppliedBiosystems website (www dot applied biosystems dot com) (FAS-Ligandassay ID: Hs_00899442_mi; IL8 assay ID: Hs_00174103_mi, TBP assay ID:Hs_00427620_mi). The relative expression mRNA level of FAS-Ligand andIL8 was computed with respect to the internal standard TBP gene tonormalize for variations in the quality of RNA and the amount of inputcDNA, as described previously (Ginestier et al. Clin. Cancer Res.12:4533-4544., herein incorporated by reference in its entirety).

Flow Cytometry Analysis.

CD44/CD24/Lin staining was performed (Ginestier et al. Cell Stem Cell1:555-567., herein incorporated by reference in its entirety). CD95/FASstaining were performed utilizing an anti-CD95 labeled APC (1:20, BDbiosciences). For CXCR1 and CXCR2 staining, primary antibodiesanti-CXCR1 (1:100, Clone 42705, R&D systems) and anti-CXCR2 (1:100,clone 48311, R&D systems) were followed by a staining with a secondaryantibody anti-mouse labeled with PE (dilution 1:250, Jackson Labs).Fresh cells were stained With 1 μg/ml PI (Sigma) for 5 min forviability.

Virus Infection.

Two different lentiviral constructs were produced for the expression ofLuciferase gene (Lenti-LUC-VSVG) (Charafe-Jauffret et al. Cancer Res.69:1302-1313., herein incorporated by reference in its entirety) and forthe inhibition PTEN expression (Lenti-PTEN-SiRNA-DsRed) (Korkaya et al.PLoS Biolog. 7:e1000121., herein incorporated by reference in itsentirety), respectively. All lentiviral constructs were prepared by theUniversity of Michigan Vector. An adenoviral construct for theoverexpression of FAK (Ad-FAK-GFP) was also utilized (Luo et al. CancerRes. 69:466-474., herein incorporated by reference in its entirety).Cells infection with different vectors was performed as previouslydescribed (Charafe-Jauffret et al. Cancer Res. 69:1302-1313., hereinincorporated by reference in its entirety). Efficiency of infection wasverified by measuring the percentage of DsRed or GFP expressing cells.

Intracardiac Inoculation.

Six weeks-old NOD/SCID mice were anesthetized with 2% isofluorane/airmixture and injected in the heart left ventricle with 250,000 cells in100 μL of sterile Dulbecco's PBS lacking Ca2+ and Mg2+. For each of thethree cell lines (HCC1954, MDA-MB-453, and SUM159) and for eachtreatment (saline or repertaxin) six animals were injected. Twelve hoursafter intracardiac injections, mice were begun on twice per dayrepertaxin injections or saline for the controls.

Bioluminescence Detection.

Baseline bioluminescence was assessed before inoculation and each weekthereafter inoculations. Bioluminescence detection procedures wasperformed as previously described (Charafe-Jauffret et al. Cancer Res.69:1302-1313., herein incorporated by reference in its entirety).Normalized photon flux represents the ratio of the photon flux detectedeach week after inoculations and the photon flux detected beforeinoculation.

CXCR1 Expression Subdivides Cancer Stem Cell Populations.

Identifying cell signaling pathways that regulate cancer stem cells(CSC) provides potential therapeutic targets in a cell population. Abreast CSC signature based on gene expression profiling that containedseveral genes potentially involved in breast CSC regulatory pathways hasbeen identified (Charafe-Jauffret et al. Cancer Res. 69:1302-1313.,herein incorporated by reference in its entirety). Among the genesoverexpressed in the breast CSC population, CXCR1 a receptor that bindsthe proinflammatory chemokine IL-8/CXCL8 appeared to be a promisingcandidate since recombinant IL-8 stimulated the self-renewal of breast C(Charafe-Jauffret et al. Cancer Res. 69:1302-1313., herein incorporatedby reference in its entirety). Utilizing flow cytometry, CXCR1 proteinexpression was measured in the breast CSC population as assessed by theALDEFLUOR assay in the human breast cancer cell lines HCC1954,MDA-MB-453, and SUM159. Cells with functional stem cell properties inNOD/SCID mouse xenographs were contained within the ALDEFLUOR-positivecell population (Charafe-Jauffret et al. Cancer Res. 69:1302-1313.,herein incorporated by reference in its entirety). The CXCR1-positivepopulation, which represents less than 2% of the total population, wasalmost exclusively contained within the ALDEFLUOR-positive population(SEE FIG. 12A and Table 4).

TABLE 4 Overlap CXCR1/ ALDEFLUOR CXCR1 ALDEFLUOR (%) (%) (%) Breastcancer cell lines HCC1954 3.42 1.72 0.94 MDA-MB-453 4.22 0.8 0.5 SUM1595.24 0.52 0.48 Human breast cancer xenografts MC1 12.3 1.81 1.32 UM2 8.41.23 0.88 UM3 9.7 0.84 0.76CXCR2 expression was also assessed. CXCR2 is a receptor that can alsobind IL-8/CXL8 although with reduced affinity compared to CXCR1. Incontrast to CXCR1-positive cells, CXCR2-positive cells were equallydistributed between the ALDEFLUOR-positive and ALDEFLUOR-negativepopulations (SEE FIG. 12A). To determine the hierarchical organizationof the cancer stem cell population according to CXCR1 expression,ALDEFLUOR-positive/CXCR1-positive and ALDEFLUOR-positive/CXCR1-negativecell populations were sorted and injected in NOD/SCID mice (SEE FIG.13). Both cell populations generated tumors. Tumor growth kineticscorrelated with the latency and size of tumor formation and the numberof cells injected. Tumors generated by theALDEFLUOR-positive/CXCR1-positive population reconstituted thephenotypic heterogeneity of the initial tumor upon serial passageswhereas the ALDEFLUOR-positive/CXCR1-negative population gave rise totumors containing only ALDEFLUOR-positive/CXCR1-negative cells. Theseresults suggest that CSC cellular hierarchy is organized according toCXCR1 expression, however both cell populations displayed similartumorigenic capacity.CXCR1 Blockade Decreases the Breast Cancer Stem Cell Population InVitro.

Three different cell lines were treated with repertaxin (100 nM), aCXCR1/2 inhibitor, to evaluate the effect of CXCR1 blockade on thebreast CSC population (Bertini et al. Proc. Natl. Acad. Sci. USA101:11791-11796., herein incorporated by reference in its entirety). ForSUM159, after three days of treatment a five-fold reduction in theproportion of ALDEFLUOR-positive cells was observed (SEE FIG. 12B). Asimilar effect was observed after treatment of SUM159 cells with ananti-CXCR1 blocking antibody. In contrast, no effect was observed aftertreatment with an anti-CXCR2 blocking antibody, suggesting that theeffects of repertaxin on the ALDEFLUOR-positive population were mediatedby CXCR1.

Data from breast tumors, as well as cell lines, demonstrate that cancerstem-like cells or cancer-initiating cells can also be isolated andpropagated as “tumorspheres” in suspension culture (Ponti et al. CancerRes. 65:5506-5511., herein incorporated by reference in its entirety).After three days of treatment with repertaxin or with the anti-CXCR1blocking antibody, when cells were detached and cultured in suspension,an 8-fold decrease in primary and secondary tumorsphere formation wasobserved compared to controls. In contrast, anti-CXCR2 blocking antibodyhad no effect on tumorsphere formation (SEE FIG. 14).

Surprisingly, after five days of treatment with repertaxin we observed amassive decrease in viability of the entire cell population as assessedby MTT assay, with only 3% of cells remaining viable (SEE FIG. 12C).Similar results were observed with the anti-CXCR1 blocking antibody butnot the anti-CXCR2 blocking antibody, thus indicating that this effectwas dependent on CXCR1 blockade. This effect of repertaxin was delayedwith loss of cell viability beginning three days after treatment (SEEFIG. 15A). Repertaxin treatment induced a similar effect on the HCC1954breast cancer cell line whereas no effect was observed on MDA-MB-453cells which harbor a PTEN mutation (Hollestelle et al. Cancer Res.5:195-201., herein incorporated by reference in its entirety) (SEE FIGS.14, 15B-C, and 16).

Utilizing a TUNEL assay, SUM159 cells were stained after 4 days oftreatment with repertaxin and a massive decrease in cell viability, dueto induction of apoptosis with 36% apoptotic cells detected afterrepertaxin treatment, was observed (SEE FIG. 12D). Results suggest thatCXCR1 blockade results in a decrease of the breast CSC populationfollowed by induction of massive apoptosis in the remaining bulk tumorpopulation.

CXCR1 Blockade Induces Cell Death in CXCR1-Negative Cells Via aBystander Effect.

The observation that repertaxin or anti-CXCR1 blocking antibody inducedmassive cell death despite the fact that the CXCR1-positive populationrepresented less than 2% of the total cell population suggested thatCXCR1 blockade in CXCR1-positive cells induced CXCR1-negative cell deathvia a bystander effect. The sorted CXCR1-positive and CXCR1-negativepopulations were treated with repertaxin (SEE FIG. 12E). Repertaxindecreased cell viability in the CXCR1-positive population within threedays whereas no effect was observed in the CXCR1-negative population.Repertaxin induced massive cell death in unseparated cells. The effectof repertaxin on cell viability of the unseparated and CXCR1-positivepopulations was dose-dependent (SEE FIG. 12E). The results areconsistent with repertaxin treatment targeting the CXCR1-positivepopulation that in turn induces CXCR1-negative cell death via abystander effect.

To determine whether this effect was mediated by a soluble factorinduced by repertaxin, conditioned medium was collected from theCXCR1-positive population after three days of repertaxin treatment anddialyzed this medium utilizing a membrane with 3.5 KDa exclusion inorder to remove repertaxin from the medium while retaining moleculeslarger than 3.5 KDa. The dialyzed conditioned medium induced a massivedecrease in cell viability in both CXCR1-negative and unseparatedpopulations but not in the CXCR1-positive population (SEE FIG. 12F).These results demonstrate that CXCR1 blockade in the CXCR1-positivepopulation induces cell death in the CXCR1-negative population via asoluble non dialyzable factor. Although the CXCR1-positive population issensitive to repertaxin it is resistant to the dialyzable death factor.

The Bystander Effect Induced by CXCR1 Blockade is Mediated byFAS-Ligand/FAS Signaling.

FAS-ligand/FAS interaction is activated in different physiologic statessuch as mammary gland involution or in conditions of tissue injuryincluding that induced by chemotherapy (Chhipa et al. J Cell Biochem.101:68-79., Song et al. J Clin. Invest 106:1209-1220, hereinincorporated by reference in their entireties). The level of solubleFAS-ligand in the medium of SUM159 cells treated with repertaxin usingan ELISA Assay to evaluate the role of FAS-ligand/FAS interaction inmediating the apoptotic bystander effect induced by CXCR1 blockade. Morethan a five-fold increase of soluble FAS-ligand in the medium of cellstreated for four days with repertaxin compared to non-treated cells wasobserved (SEE FIG. 17A). The transcriptional regulation of FAS-ligand byrepertaxin treatment by measuring FAS-ligand mRNA level was confirmed byRT-PCR (SEE FIG. 17B). A 4-fold increase of the FAS-ligand mRNA level inthe repertaxin treated cells was observed compared to non-treated cells.Similar results were observed after treatment with a FAS agonist thatactivates FAS signaling, indicating that FAS-ligand is a target of FASsignaling generating a positive feed-back loop. 100% of the SUM159 cellsexpressed FAS protein as determined by flow cytometry. Treatment of theSUM159 cells with the FAS agonist reproduced the killing effect observedwith the repertaxin treatment with massive reduction in cell viability(SEE FIG. 17C). The effect of repertaxin treatment on cell viability waspartially reversed by an anti-FAS-Ligand blocking antibody, with 44% ofcells remaining viable after treatment with repertaxin andanti-FAS-ligand antibody compared to only 3% with repertaxin alone (SEEFIG. 17C). Results suggest that the massive cell death induced byrepertaxin is due to a bystander effect mediated by the FAS-Ligand/FASpathway.

Treatment of SUM159 cells with the FAS agonist resulted in a ten-foldand three-fold increase in the percent of CXCR1-positive andALDEFLUOR-positive cells, respectively (SEE FIG. 17D/E and 18). Theeffects of repertaxin on both populations were not rescued byanti-FAS-ligand (SEE FIG. 17D/E), suggesting that the ALDEFLUOR-positivepopulation that contains the CXCR1-positive population, while directlysensitive to CXCR1 blockade which in turn induces FAS-ligand productionby these cells is resistant to FAS-ligand/FAS pro-apoptotic signaling.In contrast, the ALDEFLUOR-negative bulk cell population does notexpress CXCR1 but is sensitive to FAS-ligand mediated cell death.

FAS-ligand/FAS signaling plays an important role during mammary glandinvolution (Song et al. J Clin. Invest 106:1209-1220, hereinincorporated by reference in its entirety). The effect of CXCR1 blockadeon human normal mammary epithelial cells obtained from reductionmammoplasties was examined. As observed in breast cancer cell lines,CXCR1-positive normal mammary cells were almost exclusively containedwithin the ALDEFLUOR-positive population (SEE FIG. 19A). To determinewhether IL-8 signaling is important in normal breast stem/progenitorfunction, normal mammary epithelial cells cultured in suspension weretreated with human recombinant IL-8 and determined its effect on the CSCpopulation as measured by the formation of mammospheres (Dontu et al.Genes Dev. 17:1253-1270, herein incorporated by reference in itsentirety). Addition of IL-8 increased the formation of primary andsecondary mammospheres in a dose-dependent manner (SEE FIG. 19B),suggesting that the IL-8/CXCR1 axis may be involved in the regulation ofnormal mammary stem/progenitor cells proliferation or self-renewal.Treatment with repertaxin or the FAS agonist had no effect on theviability of normal mammary epithelial cells cultured in adherentconditions, even when high concentrations of repertaxin (500 nM) wereutilized (SEE FIG. 16A). However, as observed for breast cancer celllines, an increase of soluble FAS-ligand was detected in the medium ofnormal mammary epithelial cells treated with repertaxin (SEE FIG. 20B).This observation may be explained by the absence of FAS expression inthe normal epithelial cells cultured under these conditions (SEE FIG.20C). This is consistent with studies that demonstrate that expressionof FAS in the mammary gland occurs only during the involution processfollowing lactation (Song et al. J Clin. Invest 106:1209-1220, hereinincorporated by reference in its entirety). In contrast to its lack ofeffect on the bulk population of normal mammary epithelial cells,repertaxin significantly decreased mammosphere formation by these cells(SEE FIG. 20C).

These results suggest that the IL-8/CXCR1 axis plays an important rolein the regulation and the survival of normal and malignant mammaryepithelial stem/progenitor cell populations. The ability to affect bulkcell populations via a FAS-ligand mediated bystander effect may relateto the level of FAS expression in these cells.

CXCR1 Blockade Effects on Cancer Stem Cells are Mediated by theFAK/AKT/FOXO3A Pathway.

CXCR1 acts through a signal transduction pathway involving thephosphorylation of the focal adhesion kinase (FAK) resulting inactivation of AKT (Waugh et al. Clin. Cancer Res. 14:6735-6741., hereinincorporated by reference in its entirety). To evaluate the impact ofCXCR1 blockade on the FAK and AKT activation the level of FAK and AKTphosphorylated proteins was measured by western blot for the threedifferent cell lines. For SUM159 and HCC1954, we detected a decrease inFAK Tyr³⁹⁷ and AKT Ser⁴⁷³ phosphorylation in cells treated withrepertaxin compared to untreated cells suggesting that repertaxineffects may be mediated by the FAK/AKT pathway (SEE FIGS. 21A and 22).The observation that MDA-MB453 is resistant to repertaxin treatment maybe explained by the presence of a PTEN mutation (919G>A) that activatesthe PI3K/AKT pathway (Hollestelle et al. Mol. Cancer Res. 5:195-201.,herein incorporated by reference in its entirety). No modification inFAK Tyr³⁹⁷ and AKT Ser⁴⁷³ phosphorylation was detected after repertaxintreatment in MDAMB453 cell line (SEE FIG. 22). To confirm a functionalrole of the FAK/AKT pathway in mediating the effects of the CXCR1blockade, two viral constructs were used, one knocking down PTENexpression via a PTEN shRNA and the other leading to FAK overexpression.PTEN, through its lipid phosphatase antagonizes PI3-K/AKT signaling(Vivanco et al. Nat. Rev. Cancer 2:489-501., herein incorporated byreference in its entirety). PTEN knockdown resulted in AKT activation asdemonstrated by an increase of AKT Ser⁴⁷³ phosphorylation (SEE FIGS. 21Aand 22). PTEN knockdown blocked the effect of repertaxin treatment onFAK and AKT activity. FAK overexpression also blocked the effects ofrepertaxin and induced an activation of FAK and AKT, measured byincreased expression of FAK Tyr³⁹⁷ and AKT Ser⁴⁷³ phosphorylation. Theseresults indicate that CXCR1 blockade effects are mediated by FAK/AKTsignaling.

Utilizing immunofluorescence staining on CXCR1-positive cells confirmedthat repertaxin treatment results in a dramatic decrease of phospho-FAKand phospho-AKT expression compared to untreated cells (SEE FIG. 21B).AKT regulates the activity of the forkhead transcription factor FOXO3Avia a phosphorylation event resulting in cytoplasmic FOXO3Asequestration (Brunet et al. Mol. Cell Biol. 21:952-965., hereinincorporated by reference in its entirety). In contrast, thenon-phosphorylated form of FOXO3A transits to the nucleus where it actsas a transcription factor that regulates the synthesis of FAS-ligand(Jonsson et al. Nat. Med. 11:666-671.), herein incorporated by referencein its entirety. Repertaxin induces cell death via a FAS-ligand mediatedbystander effect; the effects of repertaxin on this signal transductionpathway were examined by immunofluorescence staining FOXO3A was presentin a cytoplasmic localization in untreated cells but shuttled to thenucleus upon repertaxin treatment (SEE FIG. 21B). This indicates thatCXCR1 blockade induces FOXO3A activity through inhibition of the FAK/AKTpathway. Cells with PTEN deletion or FAK overexpression display a highlevel of phospho-FAK and phospho-Akt expression, detected byimmunofluorescence, in both repertaxin-treated and untreated cells.Repertaxin treatment did not induce FOXO3A activation in cells with PTENdeletion or FAK overexpression, as shown by the cytoplasmic location ofFOXO3A (SEE FIG. 21B).

As a consequence of the constitutive activation of the FAK/AKT pathway,cells with PTEN deletion or FAK overexpression displayed resistance torepertaxin treatment. Cells with PTEN deletion or FAK overexpression didnot display any decrease in cell viability with repertaxin treatment. Ithas been proposed that AKT signaling plays a critical role in thebiology of CSC (SEE FIGS. 21B and 22) (Dubrovska et al. Proc. Natl.Acad. Sci. U.SA 106:268-273., Korkaya et al. PLoS Biolog. 7:e1000121.,Yilmaz et al. Nature 441:475-482., herein incorporated by reference intheir entireties). Activation of the FAK/AKT pathway blocked therepertaxin effects on the CSC populations, as shown by the maintenanceof the ALDEFLUOR-positive populations after treatment with the inhibitor(SEE FIG. 21B). All the results indicate CXCR1 blockade directly affectsthe FAK/AKT/FOXO3A pathway. Repertaxin treatment inhibits AKT signalingwhich is crucial for CSC activity and subsequently induces a bystandereffect on the bulk tumor cells mediated by CSC-generated FAS-ligand.

Repertaxin Treatment Reduces the Breast Cancer Stem Cell Population InVivo.

Recent evidence suggests that breast CSC are relatively resistant tochemotherapy and radiation and may contribute to tumor regrowthfollowing therapy (Phillips et al. J Natl. Cancer Inst. 98:1777-1785.,Yu et al. Cell 131:1109-1123., Li et al. J Natl. Cancer Inst.100:672-679., herein incorporated by reference in their entireties). TheCSC concept suggests that significant improvements in clinical outcomewill require effective targeting of the CSC population (Reya et al.Nature 414:105-111., herein incorporated by reference in its entirety).Several factors are synthesized and secreted during the apoptoticprocess when the bulk tumor cells are targeted by chemotherapy. Amongthese factors, FAS-ligand amplifies chemotherapy effects by mediating abystander killing effect (Chhipa et al. J Cell Biochem. 101:68-79.herein incorporated by reference in its entirety). Chemotherapy may alsoinduce IL-8 production in injured cells. The commonly utilizedchemotherapeutic agent, docetaxel, induced both IL-8 and FAS-ligand mRNAin SUM159 cells (SEE FIG. 10a /B). We also detected a 4-fold increase ofIL-8 mRNA level after FAS agonist treatment (SEE FIG. 10B). We haveshown that IL-8 is able to regulate the CSC population. This indicatesthat the addition of repertaxin to cytotoxic chemotherapy may block thiseffect and target the cancer stem cell population.

The SUM159 cell line and three primary human breast cancer xenograftsgenerated from three different patients (MC1, UM2, UM3) were used toexplore the efficiency of repertaxin treatment on tumor growth. Cellsfrom these tumors were transplanted orthotopically into the humanizedcleared fat-pad of NOD/SCID mice, without cultivation in vitro. For eachof these xenotransplants the CSC population was exclusively containedwithin the ALDEFLUOR-positive population (Ginestier et al. Cell StemCell 1:555-567., Charafe-Jauffret et al. Cancer Res. 69:1302-1313.,herein incorporated by reference in their entireties). In each of thetumors, the CXCR1-positive population was almost exclusively containedwithin this ALDEFLUOR-positive population (SEE Table 5) and thePTEN/FAK/AKT pathway is activated (SEE FIG. 25).

TABLE 5 Overlap CXCR1/ ALDEFLUOR CXCR1 ALDEFLUOR (%) (%) (%) Breastcancer cell lines HCC1954 3.42 1.72 0.94 MDA-MB-453 4.22 0.8 0.5 SUM1595.24 0.52 0.48 Human breast cancer xenografts MC1 12.3 1.81 1.32 UM2 8.41.23 0.88 UM3 9.7 0.84 0.7650,000 cells from each xenotransplant were injected into the humanizedfat pad of NOD/SCID mice and monitored tumor growth. When the tumor sizewas approximately 4 mm, treatment was initiated with repertaxin alone(15 mg/Kg, twice a day, during 28 days), docetaxel alone (10 mg/Kg, oncea week, during 4 weeks), or a combination of both drugs. Tumor growthwas compared to saline injected controls. For each xenotransplant, asignificant inhibition of tumor growth induced by docetaxel treatment orthe combination repertaxin/docetaxel was observed (SEE FIGS. 26A and27). Repertaxin treatment alone had a moderate impact on tumor growth.After four weeks of treatment, animals were sacrificed and the residualtumors were analyzed utilizing the ALDEFLUOR assay. Residual tumorstreated with docetaxel alone contained either an unchanged or increasedpercent of ALDEFLUOR-positive cells compared to untreated controls (SEEFIGS. 26B and 27). In contrast, repertaxin treatment alone or incombination with docetaxel reduced the ALDEFLUOR-positive population byover 75% (SEE FIGS. 26B and 27). The results were confirmed byimmunohistochemistry of ALDH1 expression in the differentxenotransplants. A decrease in ALDH1-positive cells was detected inrepertaxin-treated tumors compared to untreated tumors, whereas thepercent of ALDH1-positive cells was unchanged or increased in tumorstreated with docetaxel alone (SEE FIG. 26D).

The presence of CD44⁺/CD24⁻ cells in these tumors was evaluated. Markershave previously been shown to be expressed in breast cancer stem cells(Al Hajj et al. Proc. Natl. Acad. Sci. U.SA 100:3983-3988., hereinincorporated by reference in its entirety). The overlap between theCD44⁺/CD24⁻ phenotype and CXCR1 expression was measured. CXCR1-positivecells were present in the CD44+/CD24− cell population and the cellpopulation expressing CD24 or CD44-negative (SEE Table 6).

TABLE 6 Human Overlap CD24−/ breast cancer CD24−/CD44+ CXCR1CD44+/CXCR1+ xenografts (%) (%) (%) MC1 6.8 1.8 0.5 UM2 3.7 1.2 0.3 UM34.8 0.8 0.2In residual tumors treated with docetaxel alone, either an unchanged orincreased percent of CD44⁺/CD24⁻ cells was observed, whereas repertaxintreatment alone or in combination with docetaxel resulted in a reductionof the CD44⁺/CD24⁻ cell population (SEE FIG. 28).

A functional in vivo assay consisting of re-implantation of cells fromtreated tumors into secondary NOD/SCID mice provided a direct testassessing the tumor-initiating and self-renewal capacity of CSCremaining after treatment. Tumor cells derived from control ordocetaxel-treated animals showed similar tumor regrowth at all dilutionsin secondary NOD/SCID mice. In contrast, repertaxin treatment with orwithout docetaxel, reduced tumor growth in secondary recipients (SEEFIG. 26C). When equal numbers of cells were injected, those fromrepertaxin-treated animals showed a 2-5-fold reduction in tumor growthcompared to cells from control or docetaxel-treated animals (SEE FIG.26C). For each xenotransplant model, 1000 or 100 tumor cells obtainedfrom animals treated with a combination of repertaxin and docetaxelfailed to form any secondary tumors in NOD/SCID mice (SEE FIG. 26C, 27,and Table 7). These studies demonstrate that repertaxin treatmentspecifically targets and reduces the CSC population.

TABLE 7 Tumors/Injections number of cells injected 10,000 5,000 2,5001,000 500 250 100 Control 6/6 2/2 — 8/8 — — 6/8 Repertaxin 4/4 2/2 2/24/8 1/3 0/2 0/9 Docetaxel 2/2 4/4 2/2 6/6 3/4 2/3 8/9 Repertaxin/ 2/23/4 2/2 1/6 1/4 0/4 0/9 DocetaxelRepertaxin Treatment Inhibits FAK/AKT Signaling and Activates FOXO3A InVivo.

The expression of phospho-FAK and phospho-AKT was examined byimmunohistochemistry in each of the xenotransplants after treatment.Membranous phospho-FAK expression was detected in 50% of cells from thecontrol and docetaxel-treated tumors whereas the phospho-FAK expressionwas abolished in the tumors treated with repertaxin alone or incombination with docetaxel (SEE FIG. 26D). Similar results were observedfor the phospho-AKT expression, with 70% of cells expressing phospho-AKTin the untreated tumors, 20% phospho-AKT-positive cells indocetaxel-treated tumors and a complete inhibition of phospho-AKTexpression in the tumors treated with repertaxin alone or in combinationwith docetaxel (SEE FIG. 26D). Nuclear FOXO3A was detected in the cellsfrom the tumors treated with docetaxel alone, repertaxin alone, and thecombination repertaxin/docetaxel. These in vivo data are consistent withthe in vitro data and confirm that repertaxin treatment inhibits FAK/AKTsignaling and activates FOXO3A.

Repertaxin Treatment Reduces the Development of Systemic Metastasis.

To determine whether repertaxin reduces systemic metastasis we infectedHCC1954, MDA-MB-453, and SUM159 breast cancer cell lines with aluciferase lentivirus reporter system and introduced the cells intoNOD/SCID mice by intracardiac injection. A suspension of 250,000 cellsfor each cell line was injected and metastasis formation was monitoredonce per week by bioluminescent imaging. Twelve hours after intracardiacinjection, mice were treated twice per day by repertaxin injection orsaline for the controls. Repertaxin treatment in mice injected withHCC1954 and SUM159 cells significantly reduced metastasis formation witha lower photon flux emission in the treated compared to the untreatedmice (SEE FIG. 29A/B). Histologic sections confirmed the presence ofmetastases at several sites in untreated animals (SEE FIG. 29D).Repertaxin treatment did not have any effect on metastasis formation inmice injected with MDA-MB-453 cells (SEE FIG. 29C). The photon fluxemission and the number of animals that developed metastasis weresimilar in both repertaxin-treated and untreated group. This result isconsistent with data that described MDA-MB-453 as a cell line resistantto repertaxin due to the presence of a PTEN mutation. These resultsindicate that CXCR1 blockade with agents such as repertaxin may be ableto reduce metastasis which is mediated by the CSC population(Charafe-Jauffret et al. Cancer Res. 69:1302-1313., herein incorporatedby reference in its entirety).

Experiments conducted during development of embodiments of the presentinvention indicate that cellular subcomponents with stem cell propertiesdrive tumor growth and metastasis Visvader et al. Nat. Rev. Cancer8:755-768., herein incorporated by reference in its entirety). By virtueof their relative resistance to current therapeutic modalities, thesecells may contribute to treatment resistance and relapse (Reya et al.Nature 414:105-111., herein incorporated by reference in its entirety).The present invention provides an approach based on blocking the CXCR1cytokine receptor, which is expressed on breast cancer stem cells, toeffectively target the cancer stem cell population and to improvetherapeutic outcome. Experiments conducted during development ofembodiments of the present invention in a number of systems havedemonstrated that cytokine networks play an important role intumorigenesis. There is evidence that several of these cytokines mayregulate stem cell behavior. IL-4 is capable of regulating self-renewalof pancreatic cancer stem cells and IL-6 of regulating cancer stem cellsin colon and breast cancer (Todaro et al. Cell Stem Cell 1:389-402.,Sansone et al. J Clin. Invest 117:3988-4002., herein incorporated byreference in their entireties). The role of IL-8 in mediating tumorinvasion and metastasis has previously been demonstrated (Waugh &Wilson. Cancer Res. 14:6735-6741., Inoue et al. Clin. Cancer Res.6:2104-2119., herein incorporated by reference in their entireties). Inaddition, IL-8 increases neural stem cell self-renewal during woundhealing in the brain (Beech et al. J Neuroimmunol. 184:198-208., hereinincorporated by reference in its entirety). Lung cancer stem cells weredescribed as expressing the chemokine receptor CXCR1 (Levina et al.PLoS. ONE. 3:e3077., herein incorporated by reference in its entirety).Experiments conducted during development of embodiments of the presentinvention demonstrated that the CXCR1-positive population is almostexclusively contained within the ALDEFLUOR-positive population in breastcancer cell lines and primary xenografts as well as in normal mammarycells. The chemokine receptor is overexpressed in ALDEFLUOR-positivebreast cancer cell populations (Charafe-Jauffret et al. Cancer Res.69:1302-1313., herein incorporated by reference in its entirety). Inbreast cancers, IL-8 is produced in the tumor microenvironment by anumber of cell types including inflammatory cells, vascular endothelialcells, tumor-associated fibroblasts and mesenchymal stem cells (Waugh etal. Clin. Cancer Res. 14:6735-6741., herein incorporated by reference inits entirety). Cytokine networks mediate interaction between these celltypes, therefore cancer stem cells can be targeted through the blockadeof the IL-8 receptor CXCR1.

Utilizing in vitro assays, it was demonstrated that CXCR1 but not CXCR2(an alternative IL-8 receptor) blockade reduced the breast cancer stemcell population. This was followed by induction of apoptosis in theentire remaining cell population, which lacks CXCR1 expression. Inaddition to CXCR1 blocking antibodies, experiments performed duringdevelopment of embodiments demonstrate that repertaxin, a CXCR1/2inhibitor, induced similar effects by targeting the CXCR1-positivepopulation. In contrast to its direct effects on the CXCR1-expressingcancer stem cell population, repertaxin had no direct effect on the bulktumor cell population that lack CXCR1 expression. This indicates thatCXCR1 blockade in CXCR1-positive cells induced cell death inCXCR1-negative cells via a bystander effect. Experiments describedherein demonstrate that the FAS-ligand/FAS pathway is the mediator ofthis bystander killing effect. This phenomenon explains the efficacy ofrepertaxin treatment in inducing massive apoptosis in the entire cellpopulation despite the fact that the CXCR1-positive populationrepresents less than 1% of the cell population. The role of FAS-ligandwas demonstrated by the effective blocking of bystander killing byanti-FAS-ligand antibody.

Experiments conducted during development of embodiments of the presentinvention indicate that similar cytokine interactions may occur intumors exposed to cytotoxic chemotherapy. Chemotherapy may directlyinduce cellular apoptosis in differentiated tumor cells as well asinducing the production of FAS-ligand by these dying cells that in turninduces apoptosis in surrounding tumor cells via a FAS mediatedbystander effect. Concomitant with the production of FAS-ligand, theseinjured cells also secrete increased levels of IL-8 in a processresembling mammary involution or wound healing. As is the case in theinvoluting mammary gland, this IL-8 may stimulate breast cancer stemcells as well as protecting them from apoptosis. This may contribute tothe relative increase in cancer stem cells observed after chemotherapyin preclinical models (4) and neo-adjuvant clinical trials (5). Theeffects of chemotherapy on apoptosis and self-renewal pathways in tumorsare shown in FIG. 30.

To determine whether CXCR1 blockade could target breast cancer stemcells in vivo, the effects of the cytotoxic agent docetaxel werecompared with repertaxin on the cancer stem cell compartment and ontumor growth in NOD/SCID mice. Docetaxel is one of the most effectivechemotherapeutic agents currently used to treat women with breastcancer. The cancer stem cell populations were assessed by the ALDEFLUORassay and by serial transplantation in NOD/SCID mice. Utilizing theseassays it was determined that chemotherapy treatment alone resulted ineither no change or a relative increase in the cancer stem cellpopulations. In contrast, repertaxin treatment alone or withchemotherapy significantly reduced the cancer stem cell population.Despite the significant reduction in the tumor-initiating populations,use of repertaxin alone did not result in significant tumor shrinkage.The combination of repertaxin plus chemotherapy resulted in significantreduction in tumor size as well as in the cancer stem cell population.Combining these agents to target both cancer stem cells and bulk tumorcell populations maximizes the efficacy of these treatments.

To elucidate the mechanism of action of repertaxin, the pathwaysdownstream from CXCR1 were analyzed. The interaction between CXCR1, FAKand AKT was confirmed. CXCR1 blockade acts specifically through FAK andAKT activation. Experiments conducted during development of embodimentsof the present invention indicate that AKT activation regulates normaland malignant breast stem cell self-renewal through phosphorylation ofGSK3β resulting in the activation of the WNT pathway (Korkaya et al.PLoS Biolog. 7:e1000121, herein incorporated by reference in itsentirety). These results indicate why cells with PTEN knockdown areresistant to repertaxin. An additional function of AKT is the regulationof cell survival through phosphorylation of the forkhead transcriptionfactor FOXO3A. AKT phosphorylation of FOXO3A results in its cytoplasmicsequestration. In contrast, it was demonstrated that CXCR1 blockadeleads to decreased AKT activation resulting in the translocation ofFOXO3A in the nucleus whence it induces a number of genes includingFAS-ligand (Jonsson et al. Nat. Med. 11:666-671., herein incorporated byreference in its entirety). FAS-ligand induced via CXCR1 blockade inturn is responsible for the observed bystander killing effects (SEE FIG.30).

In addition to its role in CXCR1 signaling, FAK mediates theinteractions of cells with extracellular matrix components throughintegrin receptors (Waugh et al. Clin. Cancer Res. 14:6735-6741., hereinincorporated by reference in its entirety). FAK signaling plays a rolein regulating the self-renewal of normal and malignant mouse mammarystem cells in transgenic models (Luo et al. Cancer Res. 69:466-474.,herein incorporated by reference in its entirety). FAK activation alsopromotes cell survival by blocking FADD and RIP-mediated apoptosis(Kurenova et al. Mol. Cell Biol. 24:4361-4371., Xu et al. J Biol. Chem.275:30597-30604., herein incorporated by reference in their entireties).This provides an explanation for the resistance of the cancer stem cellpopulation to the FAS/FAS-ligand induced apoptosis.

It has been demonstrated that breast cancer stem cells play an importantrole in tumor invasion and metastasis (Croker et al. J Cell Mol. Med.2008, Charafe-Jauffret et al. Cancer Res. 69:1302-1313., hereinincorporated by reference in their entireties). It is shown herein thatIL-8 and CXCR1 also play important roles in these processes. The effectsof CXCR1 blockade was analyzed utilizing repertaxin on the formation ofexperimental metastasis. It was demonstrated that CXCR1 blockade reducesthe development of metastasis when administered subsequent tointracardiac injection of breast cancer cells.

Clinical studies utilizing repertaxin have demonstrated a lack oftoxicity. Strategies aimed at interfering with cytokine regulatory loopssuch as IL-8 and CXCR1 represent methods to target breast cancer stemcells.

REFERENCES

The following references are herein incorporated by reference in theirentireties, as if fully set forth herein.

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All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope of the presentinvention.

We claim:
 1. A method of treating a human subject diagnosed with ER-negative breast cancer, comprising: (a) administering a pharmaceutical composition containing an effective concentration of Compound 1 to the human subject in need of said treatment, wherein Compound 1 is:

and (b) administering an effective concentration of paclitaxel to the subject in need of said treatment.
 2. The method of claim 1 in which Compound 1 is administered at a dose between 3 and 60 mg per kg.
 3. A method of treating a human subject diagnosed with metastatic ER-negative breast cancer, comprising: (a) administering a pharmaceutical composition containing an effective concentration of Compound 1 to the human subject in need of said treatment, wherein Compound 1 is:

and (b) administering an effective concentration of paclitaxel to the human subject in need thereof.
 4. The method of claim 3, in which Compound 1 is administered at a dose between 3 and 60 mg per kg.
 5. The method of claim 1, wherein Compound 1 is administered concurrently with paclitaxel.
 6. The method of claim 1, wherein Compound 1 is administered at a time prior to the administration of paclitaxel.
 7. The method of claim 1, wherein Compound 1 is administered at a time subsequent to the administration of paclitaxel.
 8. The method of claim 3, wherein Compound 1 is administered concurrently with paclitaxel.
 9. The method of claim 3, wherein Compound 1 is administered at a time prior to the administration of paclitaxel.
 10. The method of claim 3, wherein Compound 1 is administered at a time subsequent to the administration of paclitaxel.
 11. The method of claim 3, wherein the method results in a reduction of metastasis.
 12. The method of claim 3, wherein Compound 1 is administered orally. 